Insulin vitamin D conjugates

ABSTRACT

The invention provides non-hormonal vitamin D conjugated to insulin peptides that result in the peptides having increased absorption, bioavailability or circulating half-life when compared to non-conjugated forms. The vitamin D targeting groups are coupled to the insulin peptides via the third carbon on the vitamin D backbone.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/919,572, filed Oct. 21, 2015 and issued as U.S. Pat. No. 9,616,109,which claims priority to U.S. Provisional Application No. 62/203,385filed Aug. 10, 2015 and U.S. Provisional Application No. 62/067,398filed Oct. 22, 2014, the contents of which are incorporated by referenceherein in their entirety.

This invention was made with Government support under Grant No.IIP-1430894 awarded by the National Science Foundation, and Grant No.W911NF-14-C-0071 awarded by the Department of Defense. The Governmenthas certain rights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 5, 2015, isnamed XTND007US1_SL.txt and is 9,002 bytes in size.

FIELD OF THE INVENTION

The invention provides non-hormonal vitamin D conjugated to insulinpeptides that result in the peptides having increased absorption,bioavailability or circulating half-life when compared to non-conjugatedforms. The vitamin D targeting groups are coupled to the insulinpeptides via the third carbon on the vitamin D backbone.

BACKGROUND OF THE INVENTION

The invention relates to improving the potency, absorption orpharmacokinetic properties of insulin peptides conjugated to certainvitamin D forms. Vitamin D plays a role in calcium, phosphate, and bonehomeostasis. The hormonal activity of vitamin D is mediated throughbinding to the vitamin D receptor (VDR). It enters the nucleus where itbinds to the vitamin D receptor element (VDRE) present in the promotersof a subset of genes that are thus responsive to hormonal vitamin D.

Vitamin D is a group of fat-soluble secosteroids. Several forms(vitamers) of vitamin D exist. The two major forms are vitamin D2 orergocalciferol, and vitamin D3 or cholecalciferol. Vitamin D without asubscript refers to vitamin D2, D3 or other forms known in the art. Inhumans, vitamin D can be ingested as cholecalciferol (vitamin D3) orergocalciferol (vitamin D2). The major source of vitamin D for mosthumans is sunlight. Once vitamin D is made in the skin or ingested, itneeds to be activated by a series of hydroxylation steps, first to25-hydroxyvitamin D (25(OH)D3) in the liver and then to1,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in the kidney. 1α,25(OH)2D3 isthe active “hormonal” form of vitamin D because it binds to VDR.25(OH)D3 is the “non-hormonal” form of vitamin D and is the majorcirculating form in the human body. It binds the vitamin D BindingProtein (DBP). It is only converted to the hormonal form as needed. Anexample of a non-hormonal vitamin D form is one that lacks a la-hydroxylgroup. Non-hormonal vitamin D forms have a greatly reduced affinity forVDR and a greatly increased affinity for DBP.

DBP is the principal transporter of vitamin D metabolites (Haddad, J.Steroid Biochem. Molec. Biol. (53)579-582 (1995)). Its concentration inthe plasma is 6-7 μM and has been detected in all fluid compartments.DBP concentrations exceed the physiological vitamin D metaboliteconcentrations. DBP is important for the translocation of vitamin D fromthe skin into circulation, and across cell membranes into the cytoplasmwhere vitamin D is activated into the hormonal form. The affinity ofnon-hormonal vitamin D for DBP is significantly higher than the affinityof the hormonal form. In contrast, the affinity of the hormonal form toVDR is significantly more than the non-hormonal form.

Vitamin D and vitamin D analogs have been approved for the treatment ofosteoporosis and secondary hyperparathyroidism. Vitamin D has also beenshown to inhibit proliferation and induce differentiation in normal aswell as cancer cells. The level of vitamin D required for this activitycauses severe toxicity in the form of hypercalcemia. Analogs of vitaminD have been approved for the treatment of psoriasis and others arecurrently being tested for cancer treatment. Many of the analogsdiscovered to have a reduced calcemic effect contain side-chainmodifications (Leyssens et al, Frontiers in Physiology 5: Article 122,1-18 (2014)). These modifications do not greatly affect VDR binding, andthus, in cell-based proliferation assays, show equal or even increasedefficacy. It was shown, however, that many of these modifications reducebinding to DBP and thereby reduce the half-life in the bloodstream.

The addition of poly(ethylene glycol) or (PEG) is a known method ofincreasing the half-life of some peptides by reducing kidney clearance,reducing aggregation, and diminishing potentially unwanted immunerecognition (Jain, Crit. Rev. Ther. Drug Carrier Syst. 25:403-447(2008)). The PEG is typically used at a considerably large size (20-40kDa) to maximize the half-life in circulation. This can be accomplishedby using either a single large PEG or multiple smaller PEGs attached tothe peptide. (Clark et al. J. Biol. Chem. 271:21969-21977 (1996);Fishburn, J. Pharm. Sci. 97:4167-4183 (2008)).

Absorption is a primary focus in drug development and medicinalchemistry because a drug must be absorbed before any medicinal effectscan take place. A drug's absorption profile can be affected by manyfactors. Additionally, the absorption properties of therapeutic peptidesvary significantly from peptide to peptide. Some therapeutic peptidesare poorly absorbed following dermal administration and cannot beadministered orally. Alternate routes of administration such asintravenous, subcutaneous, or intramuscular injections are routinelyused for some of peptides; however, these routes often result in slowabsorption and exposure of the therapeutic peptides to enzymes that candegrade them, thus requiring much higher doses to achieve efficacy.

A number of peptides have been identified as therapeutically promising.The chemical and biological properties of peptides and proteins makethem attractive candidates for use as therapeutics. Peptides andproteins are naturally-occurring molecules made up of amino acids andare involved in numerous physiological processes. Peptides and proteinsdisplay a high degree of selectivity and potency, and may not sufferfrom potential adverse drug-drug interactions or other negative sideeffects. Thus peptides and proteins hold great promise as a highlydiverse, highly potent, and highly selective class of therapeutics withlow toxicity. Peptides and proteins, however, may have short in vivohalf-lives. For such peptides, this may be a few minutes. This mayrender them generally impractical, in their native form (also referredto as “wild”, “wild type” or “wt” herein), for therapeuticadministration. Additionally, peptides may have a short duration ofaction or poor bioavailability.

Insulin is a peptide hormone produced by beta cells in the pancreas thatregulates the metabolism of carbohydrates and fats (SEQ ID NO:1 and 2).The human insulin protein is composed of 51 amino acids, and has amolecular weight of 5808 Daltons. It is a dimer of an A-chain and aB-chain that are linked by disulfide bonds. It promotes the absorptionof glucose from the blood to skeletal muscles and fat tissue and causesfat to be stored rather than used for energy. In some preferredembodiments, insulin derivatives are conjugated to the carriers of theinvention. In more preferred embodiments, the A-chain is modified atresidue 21 where the asparagine is replaced with a glycine. In otherpreferred embodiments, the B-chain is modified at position 3 (theasparagine is replaced with a lysine), position 28 (proline is replacedwith aspartic acid), positions 28 and 29 (the proline and lysine areswapped), position 29 (lysine is replaced with aspartic acid), or at thecarboxyl terminus (addition of residues that may include 1, 2, or morearginine residues).

Under normal physiological conditions, insulin is produced at a constantproportion to remove excess glucose from the blood. When control ofinsulin levels fails, however, diabetes mellitus can result. Thus,diabetic patients often receive injected insulin. Patients with type 1diabetes depend on external insulin for their survival because thehormone is no longer sufficiently produced internally. Insulin is mostcommonly injected subcutaneously. Patients with type 2 diabetes areoften insulin resistant and may suffer from an “apparent” insulindeficiency.

SUMMARY OF THE INVENTION

The invention provides carrier-drug conjugates comprising a targetinggroup that is non-hormonal vitamin D, an analog, or metabolite thereoflinked at the carbon 3 position to insulin. In some embodiments, thenon-hormonal vitamin D molecules are not hydroxylated at the carbon 1position. The carriers enhance the absorption, stability, half-life,duration of effect, potency, or bioavailability of insulin. Optionally,the carriers further comprise scaffolding moieties that arenon-releasable such as PEG and others described in this disclosure.

Thus, the invention provides a carrier-drug conjugate comprising atargeting group that is a non-hormonal vitamin D, analog, or metabolitethereof conjugated to an insulin peptide at the carbon 3 position of thenon-hormonal vitamin D targeting group. In a preferred embodiment, thenon-hormonal vitamin D is not hydroxylated at the carbon 1 position. Inanother preferred embodiment, the targeting group is conjugated to theinsulin peptide via a scaffold that is selected from the groupconsisting of poly(ethylene glycol), polylysine, polyethyleneimine,poly(propyleneglycol), a peptide, serum albumin, thioredoxin, animmunoglobulin, an amino acid, a nucleic acid, a glycan, a modifyinggroup that contains a reactive linker, a water-soluble polymer, a smallcarbon chain linker, and an additional therapeutic peptide.

The invention provides a pharmaceutical composition comprising acarrier-drug conjugate comprising a targeting group that is anon-hormonal vitamin D, analog, or metabolite thereof conjugated via ascaffold at the carbon 3 position to an insulin peptide having an aminoacid sequence with at least a 90% sequence identity to SEQ ID NO:1, 2,or 5-8. In a preferred embodiment, the carrier increases the absorption,bioavailability, or half-life of said insulin peptide in circulation. Inanother preferred embodiment, the non-hormonal vitamin D is nothydroxylated at the carbon 1 position.

In another embodiment, the scaffold is selected from the groupconsisting of poly(ethylene glycol), polylysine, polyethyleneimine,poly(propyleneglycol), a peptide, serum albumin, thioredoxin, animmunoglobulin, an amino acid, a nucleic acid, a glycan, a modifyinggroup that contains a reactive linker, a water-soluble polymer, a smallcarbon chain linker, and an additional therapeutic peptide. In a mostpreferred embodiment, the scaffold is poly(ethylene glycol).

The invention provides a method of treating a patient in need of aninsulin peptide, comprising administering an effective amount of any ofthe pharmaceutical compositions described herein. In a preferredembodiment, the pharmaceutical composition is delivered to said patientby a transdermal, oral, parenteral, subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intrasynovial, intrasternal,intrathecal, intralesional, intracranial injection, infusion,inhalation, ocular, topical, rectal, nasal, buccal, sublingual, vaginal,or implanted reservoir mode.

The invention provides the use of any of the pharmaceutical compositionsdescribed herein for the manufacture of a medicament for the treatmentof a patient in need of said medicament.

The invention provides a method of manufacturing any of thepharmaceutical compositions described herein comprising conjugating atargeting group and an insulin peptide, wherein the conjugating steputilizes a coupling group. In some embodiments, the coupling group isselected from the group consisting of an amine-reactive group, athiol-reactive group, a maleimide group, a thiol group, an aldehydegroup, an NHS-ester group, a haloacetyl group, an iodoacetyl group, abromoacetyl groups, a SMCC group, a sulfo SMCC group, a carbodiimidegroup, bifunctional cross-linkers, NHS-maleimido, and combinationsthereof. In other embodiments, the invention provides pharmaceuticalcompositions resulting from the method described herein, wherein thecompositions comprise a carrier-drug compound containing a linkageselected from the group consisting of a thiol linkage, an amide linkage,an oxime linkage, a hydrazone linkage, and a thiazolidinone linkage. Inanother embodiment, the conjugating step is accomplished bycycloaddition reactions.

The invention provides a pharmaceutical carrier comprising a formula I:B-(L)^(a)-S-(M)^(b)-C  IWherein:

-   B is a targeting group that is a non-hormonal vitamin D, analog, or    metabolite thereof conjugated at the carbon 3 position to L¹;-   S is a scaffold moiety, comprising poly(ethylene glycol),    polylysine, polyethyleneimine, poly(propyleneglycol), a peptide,    serum albumin, thioredoxin, an immunoglobulin, an amino acid, a    nucleic acid, a glycan, a modifying group that contains a reactive    linker, polylactic acid, a water-soluble polymer, a small carbon    chain linker, or an additional therapeutic moiety;-   C is an amine-reactive group, a thiol-reactive group, a maleimide    group, a thiol group, a disulfide group, an aldehyde group, an    NHS-ester group, a 4-nitrophenyl ester, an acylimidazole, a    haloacetyl group, an iodoacetyl group, a bromoacetyl groups, a SMCC    group, a sulfo SMCC group, a carbodiimide group and bifunctional    cross-linkers such as NHS-Maleimido or combinations thereof;-   L¹ and L² are linkers independently selected from —(CH₂)_(n)—,    —C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—,    —S(O)₂— and —NH—;-   L³ is —(CH₂)_(o)—;-   n is an integer from 0-3; and-   o is an integer from 0-3.

In a preferred embodiment, the pharmaceutical carrier comprises formulaV:

In another preferred embodiment, the pharmaceutical carrier comprisesformula VI:

In another preferred embodiment, the pharmaceutical carrier comprisesformula VII:

The invention provides a pharmaceutical composition, comprising aninsulin peptide, a stably attached scaffold, a targeting group that is anon-hormonal vitamin D, analog, or metabolite thereof conjugated at thecarbon 3 position, wherein after administration to a first test subject,the insulin peptide has a half-life measured by ELISA or other analysesof blood samples taken at a plurality of time points that is greaterthan a half-life of said insulin peptide administered to a second testsubject without said stably attached scaffold moiety and targeting groupas measured by the ELISA or other analyses of blood samples taken atsaid plurality of time points. In a preferred embodiment, theadministration to said first and second subjects is accomplished bysubcutaneous injection.

In another preferred embodiment, the insulin peptide stably attached tothe scaffold and targeting group retains substantially the same activityas the insulin peptide not stably attached to said scaffold andtargeting group as measured by a functional assay.

In another embodiment, a scaffold mass range is selected from the groupconsisting of about 100 Da. to 20,000 Da., 200 Da. to 15,000 Da., 300Da. to 10,000 Da., 400 Da. to 9,000 Da., 500 Da. to 5,000 Da., 600 Da.to 2,000 Da., 1000 Da. to 200,000 Da., 20,00 Da. to 200,000 Da., 100,000to 200,000 Da., 5000 Da. to 100,000 Da., 10,000 Da. to 80,000 Da.,20,000 Da. to 60,000 Da., and 20,000 Da. to 40,000 Da. In preferredembodiments, the scaffold mass is about 0.01, 0.02, 0.03, 0.04, 0.05,0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 KDa.In another preferred embodiment, the scaffold is approximately the samemass as the insulin peptide.

The invention provides a carrier-drug conjugate comprising a targetinggroup that is vitamin D, an analog, or a metabolite thereofnon-releasably conjugated to an insulin peptide. In a preferredembodiment, the vitamin D is non-hormonal. In a more preferredembodiment, the non-hormonal vitamin D is not hydroxylated at the carbon1 position. In another preferred embodiment, the insulin peptide isconjugated at the carbon 3 position of said non-hormonal vitamin Dtargeting group. In another preferred embodiment, the insulin peptideretains substantially the same activity as said insulin peptide notconjugated to said targeting group as measured by a functional assay. Inanother preferred embodiment, the targeting group is conjugated to theinsulin peptide via a scaffold that is selected from the groupconsisting of poly(ethylene glycol), polylysine, polyethyleneimine,poly(propyleneglycol), a peptide, serum albumin, thioredoxin, animmunoglobulin, an amino acid, a nucleic acid, a glycan, a modifyinggroup that contains a reactive linker, a water-soluble polymer, a smallcarbon chain linker, and an additional therapeutic peptide. In a morepreferred embodiment, the scaffold is approximately the same mass as theinsulin peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Reaction scheme showing the chemical structure and synthesesused to generate a carrier, a vitamin D-(3)-PEG_(2k)-aldehyde adduct.The carrier was generated by conjugating 1) a vitamin D analog, 2) a PEGscaffold, and 3) an aldehyde coupling group.

FIG. 2: Reaction scheme showing the chemical structure and synthesesused to generate a carrier, a vitamin D-(3)-PEG_(2k)-maleimide adduct.The carrier was generated by conjugating 1) a vitamin D analog, 2) a PEGscaffold, and 3) a maleimide coupling group.

FIG. 3: Reaction scheme showing the chemical structure and synthesesused to generate a carrier, a vitamin D-(3)-PEG_(1.3k)-NHS adduct. Thecarrier was generated by conjugating 1) a vitamin D analog, 2) a PEGscaffold, and 3) an NHS coupling group.

FIG. 4: Determination of the bioactivity of insulin conjugates comparedto unconjugated insulin at the insulin receptor, isoform B (INSRb).

FIGS. 5A-5B: Determination of the pharmacokinetics of insulin conjugatescompared to unconjugated insulin upon intravenous (FIG. 5A) andsubcutaneous (FIG. 5B) injection.

FIG. 6: Blood glucose lowering pharmacodynamics of insulin conjugatescompared to native insulin upon intravenous administration at time=0 hr.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides carrier-insulin conjugates comprising targetinggroups that are non-hormonal vitamin D, vitamin D analogs, or vitamin Dmetabolites. Examples include vitamin D-based molecules that are nothydroxylated at the carbon 1 (C1) position. The carriers are linked toinsulin peptides at the carbon 3 (C3) position. As disclosed herein,carrier groups are surprisingly effective when non-hormonal vitamin Dforms are used and the insulin peptide is linked to the carbon 3position. While not wishing to be bound by theory, it is believed thatthe hormonal forms of vitamin D are not appropriate for the carriersdescribed herein because they can be toxic due to the induction ofhypercalcemia. Also, because the hormonal forms bind the vitamin Dreceptor in cells, they may improperly target the carrier-drugconjugates to undesired cells or tissues. In contrast, non-hormonalvitamin D forms bind the Vitamin D Binding Protein (DBP) and remain incirculation longer.

The carrier molecules are attached to the insulin peptides usingchemistries described herein, described in WO2013172967, incorporatedherein in its entirety, or that are otherwise known in the art. Thecarriers improve the potency, absorption, bioavailability, circulatinghalf-life or pharmacokinetic properties of the insulin peptides. Incertain embodiments, the carriers further comprise what will bedescribed herein as a “scaffold” that acts, among other things, as anon-releasable “spacer” between the targeting group and the insulinpeptide. In other embodiments, the carriers lack a scaffold.

The carriers are designed to be suitable for use in humans and animals.The carriers serve the purpose of improving the pharmacokineticproperties of a biological or chemical entity that is coupled,conjugated, or fused to the carrier. This occurs through the interactionof the targeting group with DBP. DBP can actively transport moleculesquickly and effectively from the site of administration to thecirculating plasma, thereby reducing exposure of the drug to degradativeenzymes. The carriers, by binding to DBP, also improve the circulatinghalf-life of the drug. This increases the potency and therapeuticefficacy of the drug by preventing kidney filtration and otherelimination processes.

In describing and claiming one or more embodiments of the presentinvention, the following terminology will be used in accordance with thedefinitions described below.

The term “absorption” is the movement of a drug into the bloodstream. Adrug needs to be introduced via some route of administration (e.g. oral,topical dermal, subcutaneous, intramuscular, or intravenous) or in aspecific dosage form such as a tablet, capsule, patch, suspension,emulsion, or liquid.

The term “bioavailability” refers to the fraction of an administereddose of unchanged drug that reaches the systemic circulation, one of theprincipal pharmacokinetic properties of drugs. When a medication isadministered intravenously, its bioavailability is 100%. When amedication is administered via other routes (such as orally), itsbioavailability generally decreases (due to incomplete absorption andfirst-pass metabolism) or may vary from patient to patient.Bioavailability is an important parameter in pharmacokinetics that isconsidered when calculating dosages for non-intravenous routes ofadministration.

“Carriers” are compounds that can be conjugated to, fused to, coupled toor formulated with insulin peptides to improve the absorption,half-life, bioavailability, pharmacokinetic or pharmacodynamicproperties of the drugs. They comprise a targeting group, a couplinggroup, and optionally, a scaffold moiety. In some embodiments, carriersmay carry insulin peptides from the site of subcutaneous injection intocirculation as well as carry the insulin peptides in circulation for anextended period of time.

An “effective amount” refers to an amount of insulin peptide that iseffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic or prophylactic result. A “therapeutically effectiveamount” of an insulin peptide may vary according to factors such as thedisease state, age, sex, and weight of the individual. A therapeuticallyeffective amount may be measured, for example, by improved survivalrate, more rapid recovery, or amelioration, improvement or eliminationof symptoms, or other acceptable biomarkers or surrogate markers. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the insulin peptide are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount of insulin peptide that is effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, but not necessarily, since aprophylactic dose is used in subjects prior to or at an earlier stage ofdisease, the prophylactically effective amount will be less than thetherapeutically effective amount.

“Half-life” is a scientific term known in the art that refers to theamount of time that elapses when half of the quantity of a test moleculeis no longer detected. An in vivo half-life refers to the time elapsedwhen half of the test molecule is no longer detectable in circulatingserum or tissues of a human or animal.

A “hormone” is a biological or chemical messenger that communicatesbetween one cell (or group of cells) to another cell. As describedherein, hormones for use in the invention may be peptides, steroids,pheromones, interleukins, lymphokines, cytokines, or members of otherhormone classes known in the art.

“Homologs” are bioactive molecules that are similar to a referencemolecule at the nucleotide sequence, peptide sequence, functional, orstructural level. Homologs may include sequence derivatives that share acertain percent identity with the reference sequence. Thus, in oneembodiment, homologous or derivative sequences share at least a 70percent sequence identity. In a preferred embodiment, homologous orderivative sequences share at least an 80 or 85 percent sequenceidentity. In a more preferred embodiment, homologous or derivativesequences share at least an 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99 percent sequence identity. Homologous or derivativenucleic acid sequences may also be defined by their ability to remainbound to a reference nucleic acid sequence under high stringencyhybridization conditions. Homologs having a structural or functionalsimilarity to a reference molecule may be chemical derivatives of thereference molecule. Methods of detecting, generating, and screening forstructural and functional homologs as well as derivatives are known inthe art.

“Hybridization” generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature that can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel et al,Current Protocols in Molecular Biology, Wiley Interscience Publishers(1995).

An “individual,” “subject” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. Mammals include, but are notlimited to, primates (including human and non-human primates) androdents (e.g., mice, hamsters, guinea pigs, and rats). In certainembodiments, a mammal is a human. A “control subject” refers to ahealthy subject who has not been diagnosed as having a disease,dysfunction, or condition that has been identified in an individual,subject, or patient. A control subject does not suffer from any sign orsymptom associated with the disease, dysfunction, or condition.

A “medicament” is an active drug that has been manufactured for thetreatment of a disease, disorder, or condition.

“Nucleic acids” are any of a group of macromolecules, either DNA, RNA,or variants thereof, that carry genetic information that may directcellular functions.

“Patient response” or “response” can be assessed using any endpointindicating a benefit to the patient, including, without limitation, (1)inhibition, to some extent, of disease progression, including slowingdown and complete arrest; (2) reduction in the number of diseaseepisodes and/or symptoms; (3) inhibition (i.e., reduction, slowing downor complete stopping) of a disease cell infiltration into adjacentperipheral organs and/or tissues; (4) inhibition (i.e. reduction,slowing down or complete stopping) of disease spread; (5) decrease of anautoimmune condition; (6) favorable change in the expression of abiomarker associated with the disorder; (7) relief, to some extent, ofone or more symptoms associated with a disorder; (8) increase in thelength of disease-free presentation following treatment; or (9)decreased mortality at a given point of time following treatment.

As used herein, the term “peptide” is any peptide comprising two or moreamino acids. The term peptide includes short peptides (e.g., peptidescomprising between 2-14 amino acids), medium length peptides (15-50) orlong chain peptides (e.g., proteins). The terms peptide, medium lengthpeptide and protein may be used interchangeably herein. As used herein,the term “peptide” is interpreted to mean a polymer composed of aminoacid residues, related naturally occurring structural variants, andsynthetic non-naturally occurring analogs thereof linked via peptidebonds, related naturally-occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic peptides can besynthesized, for example, using an automated peptide synthesizer.Peptides can also be synthesized by other means such as by cells,bacteria, yeast or other living organisms. Peptides may contain aminoacids other than the 20 gene-encoded amino acids. Peptides include thosemodified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, and are well-known to those of skill in theart. Modifications occur anywhere in a peptide, including the peptidebackbone, the amino acid side chains, and the amino or carboxyl termini.

As used herein, a “pharmaceutically acceptable carrier” or “therapeuticeffective carrier” is aqueous or nonaqueous (solid), for examplealcoholic or oleaginous, or a mixture thereof, and can contain asurfactant, emollient, lubricant, stabilizer, dye, perfume,preservative, acid or base for adjustment of pH, a solvent, emulsifier,gelling agent, moisturizer, stabilizer, wetting agent, time releaseagent, humectant, or other component commonly included in a particularform of pharmaceutical composition. Pharmaceutically acceptable carriersare well known in the art and include, for example, aqueous solutionssuch as water or physiologically buffered saline or other solvents orvehicles such as glycols, glycerol, and oils such as olive oil orinjectable organic esters. A pharmaceutically acceptable carrier cancontain physiologically acceptable compounds that act, for example, tostabilize or to increase the absorption of specific inhibitor, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants such as ascorbic acid or glutathione, chelating agents, lowmolecular weight proteins or other stabilizers or excipients.

The term “pharmacokinetics” is defined as the time course of theabsorption, distribution, metabolism, and excretion of an insulinpeptide. Improved “pharmacokinetic properties” are defined as: improvingone or more of the pharmacokinetic properties as desired for aparticular therapeutic peptide. Examples include but are not limited to:reducing elimination through metabolism or secretion, increasing drugabsorption, increasing half-life, and/or increasing bioavailability.

“Scaffolds” are molecules to which other molecules can be covalently ornon-covalently attached or formulated. The scaffolds of the inventionmay act as “spacers” between the targeting group and the drug. Spacersare molecular entities that provide physical distance between the twodistinct molecular entities. Scaffolds may also contain a reactive“linker” or may have beneficial therapeutic properties in addition tothe drug. Linkers are the sites of attachment from one molecular entityto another. Thus, the scaffolds of the invention may be, for example,PEG; serum albumin, thioredoxin, an immunoglobulin, a modifying groupthat contains a reactive linker, a water-soluble polymer, or atherapeutic compound. The scaffolds and linkers of the invention arestable (i.e. non-releasable). Non-releasable linkers have more stablechemical bonds than releasable linkers to allow the attached molecularentities to remain attached in vivo. In certain embodiments, however,they may be “releasable” under specific conditions. Releasable linkershave inherent instability and allow for the release of the attachedmolecules under certain conditions over time.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.

“Stringent conditions” or “high stringency conditions”, as definedherein, can be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)overnight hybridization in a solution that employs 50% formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μl/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with a 10minute wash at 42° C. in 0.2×SSC (sodium chloride/sodium citrate)followed by a 10 minute high-stringency wash consisting of 0.1×SSCcontaining EDTA at 55° C.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed before or during the course of clinicalpathology. Desirable effects of treatment include preventing theoccurrence or recurrence of a disease or a condition or symptom thereof,alleviating a condition or symptom of the disease, diminishing anydirect or indirect pathological consequences of the disease, decreasingthe rate of disease progression, ameliorating or palliating the diseasestate, and achieving remission or improved prognosis. In someembodiments, methods and compositions of the invention are useful inattempts to delay development of a disease or disorder.

A “vitamin” is a recognized term in the art and is defined as afat-soluble or water-soluble organic substance essential in minuteamounts for normal growth and activity of the body and is obtainednaturally from plant and animal foods or supplements.

“Vitamin D” is a group of fat-soluble secosteroids. Several forms(vitamers) of vitamin D exist. The two major forms are vitamin D2 orergocalciferol, and vitamin D3 or cholecalciferol. Vitamin D without asubscript refers to vitamin D2, D3 or other forms known in the art. Inhumans, vitamin D can be ingested as cholecalciferol (vitamin D3) orergocalciferol (vitamin D2). Additionally, humans can synthesize it fromcholesterol when sun exposure is adequate. Cholecalciferol may bemodified in the liver or in vitro to 25-hydroxycholecalciferol(“25-hydroxy vitamin D”). In the kidney or in vitro, 25-hydroxy vitaminD can be modified into the distinct hormonal form of 1, 25-hydroxyvitamin D.

“Vitamin D binding protein” or “DBP” is a naturally circulating serumprotein found in all mammals that, among other activities, can bind toand transport vitamin D and its analogs to sites in the liver and kidneywhere the vitamin is modified to its active form, and it retains vitaminD in its various forms in circulation for, on average, 30 days inhumans. A DBP protein sequence is disclosed in SEQ ID NO:3 and anexemplary nucleic acid sequence encoding the DBP protein sequence isdisclosed in SEQ ID NO:4. DBP has multiple naturally-occurring isoforms.Exemplary isoforms are available in the public sequence databases (e.g.Accession Nos. NM_001204306.1, NM_001204307.1, NM_000583.3, BC036003.1,M12654.1, X03178.1, AK223458, P_001191235.1, NP_000574.2, AAA61704.1,AAD13872.1, NP_001191236.1, AAA19662.2, 154269, P02774.1, EAX05645.1,AAH57228.1, AAA52173.1, AAB29423.1, AAD14249.1, AAD14250.1, andBAD97178.1).

The invention contemplates non-hormonal vitamin D conjugates that bindDBP or functional DBP variants and homologs that contain conservative ornon-conservative amino acid substitutions that substantially retain DBPactivity. DBP binding molecules or functional DBP variants may beidentified using known techniques and characterized using known methods(Bouillon et al., J. Bone Miner Res. 6(10):1051-7 (1991), Teegarden et.al., Anal. Biochemistry 199(2):293-299 (1991), McLeod et al, J. BiolChem. 264(2):1260-7 (1989), Revelle et al., J. Steroid Biochem.22:469-474 (1985)). The foregoing references are incorporated byreference herein in their entirety.

The term “water-soluble” refers to moieties that have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like.

The invention provides effective routes for administering insulin. Inpreferred embodiments, the invention provides effective routes of drugadministration via transdermal, oral, parenteral, subcutaneous,intracutaneous, intravenous, intramuscular, intraarticular,intrasynovial, intrasternal, intrathecal, intralesional, intracranialinjection, infusion, inhalation, ocular, topical, rectal, nasal, buccal,sublingual, vaginal, or implanted reservoir modes. The preferred routeof administration is via subcutaneous injection.

In addition, the inventions described herein provide compositions andmethods for maintaining target binding activity, i.e. pharmacodynamics(PD), for insulin peptides. It further provides compositions and methodsfor improving the pharmacokinetic (PK) profiles of insulin peptides asdescribed herein. The invention further provides compositions andmethods for improved drug absorption profiles as compared to the drugabsorption profiles for the drugs using the same routes ofadministration or different routes of administration but without theinventions described herein. The invention further provides compositionsand methods for improved drug bioavailability profiles as compared tothe drug bioavailability profiles for the drugs using the same routes ofadministration or different routes of administration but without thecarriers described herein. The invention further provides compositionsand methods for improved drug half-life profiles as compared to the drughalf-life profiles for the drugs using the same routes of administrationor different routes of administration but without the inventionsdescribed herein.

The non-hormonal vitamin D carriers disclosed herein may improve theabsorption, half-life, bioavailability, or pharmacokinetic properties ofthe linked insulin peptides. While not wishing to be bound by theory,the carriers have the properties of binding to the body's natural DBP.DBP may transport the carrier-drug complex from the site ofadministration to the circulating serum. The vitamin D-DBP interactionmay retain the insulin peptides in circulation for an extended period oftime. This can prevent its excretion from the body and increase theexposure of the insulin peptide in the body to achieve a longer lastingtherapeutic effect. Additionally, a smaller dose of drug may be requiredwhen conjugated the carrier when compared to the unmodified form.

The insulin peptide carrier conjugates of the invention typically haveabout 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting groups individuallyattached to an insulin peptide. The structure of each of the targetinggroups attached to the insulin peptide may be the same or different. Inpreferred embodiments, one or more targeting groups are stably ornon-releasably attached to the insulin peptide at the N-terminus,C-terminus, or other portion of an insulin peptide. For example, aninsulin peptide carrier conjugate may comprise a targeting groupattached to the N-terminus and additionally a targeting group attachedto a lysine residue. In another embodiment, an insulin peptide carrierconjugate has a targeting group attached to an insulin peptide via amodification such as a sugar residue as part of a glycosylation site, oron an acylation site of a peptide or attached to a phosphorylation siteor other natural or non-natural modifications that are familiar to oneskilled in the art. Also contemplated are attachment sites using acombination of sites mentioned above. One preferred embodiment of thepresent invention comprises a targeting group that is attached to theinsulin peptide at one specific site on the peptide. In anotherpreferred embodiment, the attachment site on a protein may be acysteine, lysine, the N-terminus or C-terminus.

In another embodiment, the scaffold is a pharmaceutically acceptablecarrier. In preferred embodiments, the scaffold is poly(ethyleneglycol), polylysine, polyethyleneimine, poly(propyleneglycol), apeptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, anucleic acid, a glycan, a modifying group that contain a reactivelinker, a water-soluble polymer, a small carbon chain linker, or anadditional therapeutic moiety.

In one embodiment, water-soluble scaffold moieties have some detectabledegree of solubility in water. Methods to detect and/or quantify watersolubility are well known in the art. Exemplary water-soluble polymersinclude peptides, saccharides, poly(ethers), poly(amines),poly(carboxylic acids) and the like.

Peptides can have mixed sequences or be composed of a single amino acid,e.g., poly(lysine). An exemplary polysaccharide is poly(sialic acid). Anexemplary poly(ether) is poly(ethylene glycol), e.g. m-PEG

Poly(ethyleneimine) is an exemplary polyamine, and poly(acrylic) acid isa representative poly(carboxylic acid). The polymer backbone of thewater-soluble polymer can be poly(ethylene glycol) (i.e. PEG). However,it should be understood that other related polymers are also suitablefor use in the practice of this invention and that the use of the termPEG or poly(ethylene glycol) is intended to be inclusive and notexclusive in this respect. The term PEG includes poly(ethylene glycol)in any of its forms, including alkoxy PEG difunctional PEG multiarmedPEG forked PEG branched PEG, pendent PEG (i.e. PEG or related polymershaving one or more functional groups pendent to the polymer backbone),or PEG with degradable linkages therein. The polymer backbone can belinear or branched.

Branched polymer backbones are generally known in the art. Typically, abranched polymer has a central branch core moiety and a plurality oflinear polymer chains linked to the central branch core. PEG is commonlyused in branched forms that can be prepared by addition of ethyleneoxide to various polyols, such as glycerol, pentaerythritol andsorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)m in which R represents thecore moiety, such as glycerol or pentaerythritol, and m represents thenumber of arms. Multi-armed PEG molecules, such as those described inU.S. Pat. No. 5,932,462, which is incorporated by reference herein inits entirety, can also be used as the polymer backbone.

Many other polymers are also suitable for the invention. Polymerbackbones that are non-peptidic and water-soluble, with from 2 to about300 termini, are particularly useful in the invention. Examples ofsuitable polymers include, but are not limited to, other poly(alkyleneglycols), such as poly(propylene glycol) (“PPG”), copolymers of ethyleneglycol and propylene glycol and the like, poly(oxyethylated polyol),poly(olefinic alcohol), polyvinylpyrrolidone), polylysine,polyethyleneimine, poly(hydroxypropylmethacrylamide), poly(α-hydroxyacid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), such as described in U.S. Pat. No.5,629,384, which is incorporated by reference herein in its entirety,and copolymers, terpolymers, and mixtures thereof. Although themolecular weight of each chain of the polymer backbone can vary, it istypically in the range of about 100 Da to about 100,000 Da.

In other embodiments, the scaffold moiety may be a peptide, serumalbumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid,a glycan, a modifying group that contains a reactive linker, awater-soluble polymer, a small carbon chain linker, or an additionaltherapeutic peptide. In one embodiment, the scaffold moieties arenon-toxic to humans and animals. In another embodiment, the scaffoldsare endogenous serum proteins. In another embodiment, the scaffoldmoieties are water-soluble polymers. In another embodiment, thescaffolds are non-naturally-occurring polymers. In another embodiment,the scaffolds are naturally-occurring moieties that are modified bycovalent attachment to additional moieties (e.g., PEG; poly(propyleneglycol), poly(aspartate), biomolecules, therapeutic moieties, ordiagnostic moieties). The scaffolds and linkers of the invention arestable (i.e. non-releasable). In certain embodiments, however, they maybe “releasable” under specific conditions.

The conjugation of hydrophilic polymers, such as PEG; is known in theart. In its most common form, PEG is a linear polymer terminated at eachend with hydroxyl groups: HO—CH2CH2O—(CH2CH2O)n-CH2CH2-OH where ntypically ranges from about 3 to about 4000. In a preferred embodiment,the PEG has a molecular weight distribution that is essentiallyhomodisperse. In another preferred embodiment, the PEG is a linearpolymer. In another preferred embodiment the PEG is a branched polymer.

Many end-functionalized or branched derivatives and various sizes areknown in the art and commercially available. By way of example,conjugation of the PEG or PEO may be carried out using the compositionsand methods described herein and in U.S. Pat. No. 7,803,777 (Defrees etal.) and U.S. Pat. No. 4,179,337 (Davis et al.), each of which areincorporated by reference herein in their entirety.

In some embodiments, smaller therapeutic peptides such as insulin arepaired with smaller scaffold moieties. It is contemplated, however, thatinsulin could be paired with a larger scaffold moiety. In someembodiments, a scaffold that is approximately equal to or smaller thanthe molecular weight of insulin results in an efficacious carrier-drugconjugate. Improvements in efficacy may be obtained by empiricallyadjusting the scaffold size further. Without wishing to be bound bytheory, the pharmacokinetic properties and efficacy of the conjugatesmay be enhanced when a scaffold (in combination with linkers as needed)is big enough to ablate potential steric hindrance of the drug by DBPbinding and vice versa. Thus, an insulin peptide is conjugated so thatits active region is exposed and available for functional activity andthe carrier is able to bind DBP. Additional embodiments providenon-releasable attachments that extend the circulation of insulinpeptides. In some embodiments, the scaffold may be selected to beapproximately equal to the peptide's molecular weight.

In preferred embodiments, the conjugation of the insulin peptide retainssubstantially all of its activity following the conjugation. The activeregion of insulin is known in the art. In other embodiments, the insulinpeptide is therapeutically active while remaining linked to the carrier.This embodiment may maximize the time in circulation and as well as itsefficacy.

The scaffolds of the present invention, for example, could have amolecular weight of 100 Daltons (Da.), 500 Da., 1000 Da., 2000 Da., 5000Da., 10,000 Da., 15,000 Da., 20,000 Da., 30,000 Da., 40,000 Da. or60,000 Da. In one embodiment of the invention, “small” scaffolds may bebetween about 100 Da. and 20,000 Da. In another embodiment, “large”scaffolds may be greater than about 20,000 Da. to about 200,000 Da. Inpreferred embodiments, the scaffold moiety is between about 100 Da. and200,000 Da. In more preferred embodiments, the scaffold is between about100 Da. and 20,000 Da., 200 Da. and 15,000 Da., 300 Da. and 10,000 Da.,400 Da. and 9,000 Da., 500 Da. and 5,000 Da., 600 Da. and 2,000 Da.,1000 Da. and 200,000 Da., 20.00 Da. and 200,000 Da., 100,000 and 200,000Da., 5000 Da. and 100,000 Da., 10,000 Da. and 80,000 Da., 20,000 Da. and60,000 Da., or 20,000 Da. and 40,000 Da. The size of the scaffolds maybe varied to maximize absorption, bioavailability, circulatinghalf-life, or efficacy of the conjugated insulin peptide.

Another component of the carrier molecule preferably comprises acoupling group that is used to covalently attach the drug to thescaffold or the carrier. The coupling groups of the invention include anamine-reactive group, a thiol-reactive group, a maleimide group, a thiolgroup, an aldehyde group, an NHS-ester group, a haloacetyl group, aniodoacetyl group, a bromoacetyl groups, a SMCC group, a sulfo SMCCgroup, a carbodiimide group and bifunctional cross-linkers such asNHS-maleimido, combinations thereof, or other coupling groups familiarto persons skilled in the art. The coupling groups of the invention canpromote thiol linkages, amide linkages, oxime linkages, hydrazonelinkages, thiazolidinone linkages or utilize cycloaddition reactionsalso called click chemistry to couple the carrier to an insulin peptide.In another embodiment, the composition preferably includes a combinationof insulin and one or more additional peptides or other moleculesattached to the coupling group of the scaffold molecule. The linkers ofthe invention may be between about 40 and 100 Daltons. In preferredembodiments, the linkers may be between about 40-50, 50-60, 60-70,70-80, 80-90, or 90-100 Daltons. The linkers may also be varied toaffect the stability or releasability of the link between the carrierand the insulin peptides or other molecules.

NHS groups are known to those skilled in the art as being useful forcoupling to native peptides and proteins without having to engineer in asite of attachment. NHS groups allow attachment to most proteins andpeptides that contain amino acids with amine groups such as a lysineresidue. Utilization of NHS groups allows for flexibility in the site ofcarrier conjugation as protein structure and reaction time can influencethe attachment site and number of carrier molecules conjugated to aninsulin peptide. By way of example, controlling the molar ratio ofNHS-carrier to a therapeutic peptide, one skilled in the art can havesome control over the number of carrier molecules attached to thetherapeutic peptide thus allowing for more than one carrier to beconjugated to a given therapeutic peptide, if desired.

Conjugation of the carrier to an insulin peptide is achieved by mixing asolution of the molecules together in a specific molar ratio usingcompatible solutions, buffers or solvents. For example, a molar ratio ofabout 1:1, 2:1, 4:1, 5:1, 10:1, 20:1, 25:1, 50:1, 100:1, 1000:1, orabout 1:2, 1:4, 1:5, 1:10, 1:20 1:25, 1:50, 1:100 or 1:1000 of carrierto an insulin peptide could be used. By varying the ratio, this couldresult in different numbers of individual carriers attached to theinsulin peptide, or could help to select a specific site of attachment.Attachment of the carriers is also pH, buffer, salt and temperaturedependent and varying these parameters among other parameters caninfluence the site of attachment, the number of carriers attached, andthe speed of the reaction. For example, by selecting a pH for thereaction at or below pH 6 could help selectively conjugate an aldehydeversion of the carrier to the N-terminus of the insulin protein orpeptide.

Additionally, in order to retain substantially the same activity of theinsulin peptides, conjugation to the carriers will be at a site on themolecules that do not interfere with insulin function. This may requireconjugation to the amino terminus, the carboxy terminus, or to aninternal reactive amino acid.

In certain embodiments, the present invention provides carriers thatinclude those of formula I:B-(L)^(a)-S-(M)^(b)-C  IWherein:

-   B is a targeting group selected from vitamin D, a vitamin D analog,    a vitamin D-related metabolite, an analog of a vitamin D    related-metabolite, a peptide that binds DBP, an anti-DBP antibody,    an anti-DBP antibody derivative, a nucleotide aptamer that binds    DBP, or a small carbon-based molecule that binds DBP;-   S is a scaffold moiety, comprising poly(ethylene glycol),    polylysine, polyethyleneimine, poly(propyleneglycol), a peptide,    serum albumin, thioredoxin, an immunoglobulin, an amino acid, a    nucleic acid, a glycan, a modifying group that contains a reactive    linker, polylactic acid, a water-soluble polymer, a small carbon    chain linker, or an additional therapeutic peptide;-   C is an amine-reactive group, a thiol-reactive group, a maleimide    group, a thiol group, a disulfide group, an aldehyde group, an    NHS-ester group, a 4-nitrophenyl ester, an acylimidazole, a    haloacetyl group, an iodoacetyl group, a bromoacetyl groups, a SMCC    group, a sulfo SMCC group, a carbodiimide group and bifunctional    cross-linkers such as NHS-maleimido or combinations thereof;-   (L)^(a) and (M)^(b) are linkers independently selected from    —(CH₂)_(n)—, —C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—,    —S(O)—, —S(O)₂— and —NH—;-   a is an integer from 0-4; and-   b is an integer from 0-4; and-   n is an integer from 0-3.

In preferred embodiments, the present invention provides carriers thatinclude those of formula I:B-(L)^(a)-S-(M)^(b)-C  IWherein:

-   B is a targeting group selected from vitamin D, a vitamin D analog,    a vitamin D-related metabolite, an analog of a vitamin D    related-metabolite, or a small carbon-based molecule that binds DBP;-   S is a scaffold moiety, comprising poly(ethylene glycol),    polylysine, poly(propyleneglycol), a peptide, serum albumin, an    amino acid, a nucleic acid, a glycan, polylactic acid, a    water-soluble polymer, or a small carbon chain linker;-   C is a maleimide group, a thiol group, a disulfide group, an    aldehyde group, an NHS-ester group, an iodoacetyl group, or a    bromoacetyl group;-   (L)^(a) and (M)^(b) are linkers independently selected from    —(CH₂)_(n)—, —C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—,    —S(O)—, —S(O)₂— and —NH—;-   a is an integer from 0-4; and-   b is an integer from 0-4; and-   n is an integer from 0-3.

In more preferred embodiments, the present invention provides carriersthat include those of formula I:B-(L)^(a)-S-(M)^(b)-C  IWherein:

-   B is a targeting group selected from vitamin D, a vitamin D analog,    or a vitamin D-related metabolite;-   S is a scaffold moiety, comprising poly(ethylene glycol), polylysine    or poly(propyleneglycol);-   C is a maleimide group, a disulfide group, an aldehyde group, an    NHS-ester group or an iodoacetyl group;-   (L)^(a) and (M)^(b) are linkers independently selected from    —(CH₂)_(n)—, —C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—,    —S(O)—, —S(O)₂— and —NH—;-   a is an integer from 0-4; and-   b is an integer from 0-4; and-   n is an integer from 0-3.

In most preferred embodiments, the present invention provides carriersthat include those of formulas IIa, IIb, and IIc:

Wherein:

-   B is a targeting group selected from vitamin D, a vitamin D analog,    or a vitamin D-related metabolite;-   S is a scaffold moiety, comprising poly(ethylene glycol), or    poly(propyleneglycol); and-   C is a maleimide group, a disulfide group, an aldehyde group, an    NHS-ester group or an iodoacetyl group;-   L¹ is —(CH₂)_(n)—;-   L³ is —(CH₂)_(o)—;-   (M)^(b) are linkers independently selected from —(CH₂)_(n)—,    —C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—, —S(O)—,    —S(O)₂— and —NH—;-   b is an integer from 0-4; and-   n is 3; and-   o is 1.

In PCT/US2013/031788, which is incorporated herein by reference,conjugation at the C25 position of 25-hydroxy-vitamin D3 is exemplified.The present invention incorporates conjugation at the C3 position of25-hydroxy-vitamin D3. This gives improved half-life extension andbioavailability compared to the C25 conjugates.

In certain most preferred embodiments of formula IIa, B is representedby formula III, S is poly(ethylene glycol) and (M)^(b)-C is representedby formula IVa.

In certain most preferred embodiments of formula IIb, B is representedby formula III, S is poly(ethylene glycol) and (M)^(b)-C is representedby formula IVb.

In certain most preferred embodiments of formula IIc, B is representedby formula III, S is poly(ethylene glycol) and (M)^(b)-C is representedby formula IVc.

In certain most preferred embodiment, S is between about 100 Da. and200,000 Da. In other most preferred embodiments, the scaffold moiety isbetween about 100 Da. and 20,000 Da., 200 Da. and 15,000 Da., 300 Da.and 10,000 Da., 400 Da. and 9,000 Da., 500 Da. and 5,000 Da., 600 Da.and 2,000 Da., 1000 Da. and 200,000 Da., 5000 Da. and 100,000 Da.,10,000 Da. and 80,000 Da., 20,000 Da. and 60,000 Da., or 20,000 Da. and40,000 Da.

In a specific embodiment, the present invention provides a carrierrepresented by formula v.

In another specific embodiment, the present invention provides a carrierrepresented by formula VI.

In another specific embodiment, the present invention provides a carrierrepresented by formula VII.

In certain embodiments, the present invention provides a method forproducing a carrier of formula I:B-(L)^(a)-S-(M)^(b)-C  Icomprising the step of reacting a compound of formula Ia:B-L¹-NH₂  Iawith a compound of formula Ib:HOOC-L³-S-(M)^(b)-C  Ibin the presence of an amide coupling agent,wherein B, S, C and L¹, L³, and (M)^(b) are defined as above and L² is—C(O)NH—.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable amide coupling agent may be used to form a compound offormula I.

Suitable amide coupling agents include, but are not limited to2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTUand T3P. In certain embodiments, the amide coupling agent is used alone.In certain embodiments, the amide coupling agent is used with aco-reagent such as HOBT or DMAP. In certain embodiments, the amidecoupling agent is used with a base such as triethylamine ordiisopropylethylamine. In certain embodiments, the amide coupling agentis used with both a co-reagent such as HOBT or DMAP and a base such astriethylamine or diisopropylethylamine. One skilled in the art willrecognize that co-reagents other than HOBT or DMAP may be used.Furthermore, one skilled in the art will recognize that bases other thantriethylamine or diisopropylethylamine may be used.

One skilled in the art will recognize that any suitable leaving groupmay be coupled with the carboxylic acid of formula Ib in the presence ofa suitable coupling agent to form an active ester of formula Ic:

wherein R is a suitable leaving group including, but are not limited toimidazole, HOBT, NHS and 4-nitrophenol. Suitable coupling reagentsinclude, but are not limited to 2-chloromethylpyridinium iodide, BOP,PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P. In some embodiments, thepresent invention provides a method for producing a carrier of formulaI:B-(L)^(a)-S-(M)^(b)-C  Icomprising the step of reacting a compound of formula Ia:B-L¹-NH₂  Iawith a compound of formula Ic:ROOC-L³-S-(M)^(b)-C  Icwherein B, S, C, R and L¹, L³, and (M)^(b) are defined as above and L²is —C(O)NH—.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, the amide coupling is performed with a base suchas triethylamine or diisopropylethylamine. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

In certain other embodiments, the present invention provides a methodfor producing a carrier of formula IIa:

comprising the steps of reacting a compound of formula Ia:B-L¹-NH₂  Iawith a compound of formula Id:HOOC-L³-S-(M)^(b)-CH₂OH  Idin the presence of an amide coupling agent forming a compound of formulaIe; and

Oxidation of the primary alcohol of formula Ie to an aldehyde of formulaIIa;

wherein B, S, L¹, L³, (M)^(b), b, n and o are defined as above and L² is—C(O)NH— and C is an aldehyde group.

Any suitable oxidizing agent may be used to form a compound of formulaIIa. Suitable oxidizing agents include, but are not limited to, theCollins reagent, PDC, PCC, oxalyl chloride/DMSO (Swern oxidation),SO₃-pyridine/DMSO (Parikh-Doehring oxidation), Dess-Martin periodinane,TPAP/NMO, and TEMPO/NaOCl.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable amide coupling agent may be used to form a compound offormula Ie. Suitable amide coupling agents include, but are not limitedto 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI,TBTU and T3P. In certain embodiments, the amide coupling agent is usedalone. In certain embodiments, the amide coupling agent is used with aco-reagent such as HOBT or DMAP. In certain embodiments, the amidecoupling agent is used with a base such as triethylamine ordiisopropylethylamine. In certain embodiments, the amide coupling agentis used with both a co-reagent such as HOBT or DMAP and a base such astriethylamine or diisopropylethylamine. One skilled in the art willrecognize that co-reagents other than HOBT or DMAP may be used.Furthermore, one skilled in the art will recognize that bases other thantriethylamine or diisopropylethylamine may be used.

In certain embodiments, any suitable leaving group can be coupled with acarboxylic acid of formula Id in the presence of a suitable couplingreagent to form an active ester of formula If:

wherein R is a suitable leaving group including, but are not limited toimidazole, HOBT, NHS and 4-nitrophenol. Suitable coupling reagentsinclude, but are not limited to 2-chloromethylpyridinium iodide, BOP,PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P.

In some embodiments, the present invention provides a method forproducing a carrier of formula Ie:

comprising the step of reacting a compound of formula Ia;B-L¹-NH₂  Iawith a compound of formula If; andROOC-L³-S-(M)^(b)-CH₂OH  If

Oxidation of the primary alcohol of formula Ie to an aldehyde of formulaIIa;

wherein B, S, C, R and L¹, L³, and (M)^(b) are defined as above and L²is —C(O)NH—.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, the amide coupling is performed with a base suchas triethylamine or diisopropylethylamine. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

Any suitable oxidizing agent may be used to form a compound of formulaIIa. Suitable oxidizing agents include, but are not limited to, theCollins reagent, PDC, PCC, oxalyl chloride/DMSO (Swern oxidation),SO₃-pyridine/DMSO (Parikh-Doehring oxidation), Dess-Martin periodinane,TPAP/NMO, and TEMPO/NaOCl.

In certain other embodiments, the present invention provides a methodfor producing a carrier of formula IIc:

comprising the steps of reacting a compound of formula Ia:B-L¹-NH₂  Iawith a compound of formula Ig:ROOC—S-(M)^(b)-COOH  Igforming a compound of formula Ih; and

Converting a carboxylic acid of formula Ih to an active ester of formulaIIc;

wherein B, S, C, R, L¹, (M)^(b), b, n and o are defined as above and L²is —C(O)NH—.

One skilled in the art will recognize that a compound of formula Ia canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable leaving group can be coupled with a carboxylic acid offormula Ih in the presence of a suitable coupling reagent to form anactive ester of formula IIc. Suitable leaving groups include, but arenot limited to imidazole, HOBT, NHS and 4-nitrophenol. Suitable couplingreagents include, but are not limited to 2-chloromethylpyridiniumiodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU and T3P.

In some embodiments, an active ester of formula IIc is formed from acarboxylic acid of formula Ih using a combination of a suitable leavinggroup and a coupling reagent.

In some embodiments, an active ester of formula IIc is formed from acarboxylic acid of formula Ih using a single reagent that produces aleaving group and also effects a coupling reaction. Such reagentsinclude, but are not limited to 1,1′-carbonyldiimidazole,N,N′-disuccinimidyl carbonate, 4-nitrophenyl trifluoroacetate and HBTU.In some embodiments, the single reagent is used alone. In otherembodiments, the single reagent is used with an acyl transfer catalyst.Such acyl transfer catalysts include, but are not limited to DMAP andpyridine. One skilled in the art will recognize that additional acyltransfer catalysts may be used.

In a specific embodiment, the present invention provides a method forproducing a carrier represented by formula V:

comprising the step of reacting a compound of formula Va:

with a compound of formula Vb:

to form a compound of formula Vc;

Reduction of the nitrile group to form the amine of formula Vd;

Reaction of the compound of formula Vd with a compound of formula Ve;

To form a compound of the formula Vf

Oxidation of the primary alcohol of formula Vf to form the aldehyde offormula V.

In some embodiments, the reaction of a compound of formula Vb with acompound of formula Va is promoted by addition of Triton B. One skilledin the art will recognize that other reagents may be used to promotenucleophilic addition to acrylonitrile.

In some embodiments, reduction of the nitrile of formula Vc to the amineof formula Vd is performed using AlCl₃/LAH. One skilled in the art willrecognize that other reduction reagents may be used including sodium,H₂/Pd, H₂/Raney nickel, and diborane.

One skilled in the art will recognize that a compound of formula Vd canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, a base such as triethylamine ordiisopropylethylamine is used to promote coupling of the NHS-ester offormula Ve with the amine of formula Vd. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

Any suitable oxidizing agent may be used to form a compound of formulaV. Suitable oxidizing agents include, but are not limited to, theCollins reagent, PDC, PCC, oxalyl chloride/DMSO (Swern oxidation),SO₃-pyridine/DMSO (Parikh-Doehring oxidation), Dess-Martin periodinane,TPAP/NMO, and TEMPO/NaOCl.

In another specific embodiment, the present invention provides a methodfor producing a carrier represented by formula VI:

comprising the steps of reacting a compound of formula Vd:

in the presence of an amide coupling agent with a compound of formulaVIa:

One skilled in the art will recognize that a compound of formula Vd canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

Any suitable amide coupling agent may be used to form a compound offormula VI. Suitable amide coupling agents include, but are not limitedto 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI,TBTU and T3P. In certain embodiments, the amide coupling agent is usedalone. In certain embodiments, the amide coupling agent is used with aco-reagent such as HOBT or DMAP. In certain embodiments, the amidecoupling agent is used with a base such as triethylamine ordiisopropylethylamine. In certain embodiments, the amide coupling agentis used with both a co-reagent such as HOBT or DMAP and a base such astriethylamine or diisopropylethylamine. One skilled in the art willrecognize that co-reagents other than HOBT or DMAP may be used.Furthermore, one skilled in the art will recognize that bases other thantriethylamine or diisopropylethylamine may be used.

In another specific embodiment, the present invention provides a methodfor producing a carrier represented by formula VII:

comprising the steps of reacting a compound of formula Vd:

with a compound of formula VIIa:

forming a compound of formula VIIb; and

Converting a carboxylic acid of formula VIIb to an active ester offormula VII;

One skilled in the art will recognize that a compound of formula Vd canbe used either as a free base or as a suitable salt form. Suitable saltforms include, but are not limited to TFA, HCl, HBr, MsOH, TfOH andAcOH.

In certain embodiments, a base such as triethylamine ordiisopropylethylamine is used to promote coupling of the NHS-ester offormula VIIa with the amine of formula Va. One skilled in the art willrecognize that bases other than triethylamine or diisopropylethylaminemay be used.

NHS can be coupled with a carboxylic acid of formula VIIb in thepresence of a suitable coupling reagent to form an active ester offormula VII. Suitable coupling reagents include, but are not limited to2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI,TBTU, and T3P.

In some embodiments, an active ester of formula VII is formed from acarboxylic acid of formula VIIb using a combination of NHS and acoupling reagent.

In some embodiments, an active ester of formula VII is formed from acarboxylic acid of formula VIIb using a single reagent that produces aleaving group and also effects a coupling reaction. Such reagentsinclude, but are not limited to, N,N′-disuccinimidyl carbonate. In someembodiments, the single reagent is used alone. In other embodiments thereagent is used with an acyl transfer catalyst. Such acyl transfercatalysts include, but are not limited to DMAP and pyridine. One skilledin the art will recognize that additional acyl transfer catalysts may beused.

One skilled in the art will recognize that there are other methods toconjugate a linker and scaffold to the C3 position of vitamin Dderivatives and analogues. For example, the C3 hydroxy group may beacylated by various groups as practiced by N. Kobayashi, K. Ueda, J.Kitahori, and K. Shimada, Steroids, 57, 488-493 (1992); J. G Haddad, etal., Biochemistry, 31, 7174-7181 (1992); A. Kutner, R. P. Link, H. K.Schnoes, H. F. DeLuca, Bioorg. Chem., 14, 134-147 (1986); and R. Ray, S.A. Holick, N. Hanafin, and M. F. Holick, Biochemistry, 25, 4729-4733(1986). The foregoing references are incorporated by reference in theirentirety. One skilled in the art will recognize that these chemistriescould be modified to synthesize compounds of the formula I:B-(L)^(a)-S-(M)^(b)-C  Iwherein B, S, C, (L)^(a), and (M)^(b) are defined as above.

If desired, insulin peptide carrier conjugates having differentmolecular weights can be isolated using gel filtration chromatographyand/or ion exchange chromatography. Gel filtration chromatography may beused to fractionate different insulin peptide carrier conjugates (e.g.,1-mer, 2-mer, 3-mer, and so forth, wherein “1-mer” indicates onetargeting group molecule per insulin peptide, “2-mer” indicates twotargeting groups attached to an insulin peptide, and so on) on the basisof their differing molecular weights (where the difference correspondsessentially to the average molecular weight of the targeting group).

Gel filtration columns suitable for carrying out this type of separationinclude Superdex and Sephadex columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) optical density (OD) at280 nm for protein content, (ii) bovine serum albumin (BSA) proteinanalysis, and (iii) sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS PAGE).

Separation of insulin peptide carrier conjugates can also be carried outby reverse phase chromatography using a reverse phase-high performanceliquid chromatography (RP-HPLC) C18 column (Amersham Biosciences orVydac) or by ion exchange chromatography using an ion exchange column,e.g., a DEAE- or CM-Sepharose ion exchange column available fromAmersham Biosciences. The resulting purified compositions are preferablysubstantially free of the non-targeting group-conjugated insulinpeptide. In addition, the compositions preferably are substantially freeof all other non-covalently attached targeting groups.

As described herein, the carriers of the invention may be non-hormonal25-hydroxy vitamin D or analogs thereof having a coupling group on the3′ carbon. “25-hydroxy vitamin D analogs” as used herein includes bothnaturally-occurring vitamin D metabolite forms as well as otherchemically-modified forms. The carriers of the invention do not includean active (i.e. hormonal) form of vitamin D (typically having a hydroxylgroup at the 1 carbon). These compounds are based on the vitamin Dstructure and retain partial function of vitamin D (i.e. they interactwith DBP), albeit at varying affinities. The following list exemplifiesvitamin D analog forms known in the art. They may, however, be hormonalor have the C1 hydroxyl group. They are presented here solely for theirchemical properties as vitamin D analogs, not for their functionalhormonal properties: OCT, a chemically synthesized of 1,25(OH)2D3 withan oxygen atom at the 22 position in the side chain (Abe et. al., FEBSLett. 226:58-62 (1987)); Gemini vitamin D analog,1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol(BXL0124) (So et al., Mol Pharmacol. 79(3):360-7 (2011)); Paricalcitol,a vitamin D2 derived sterol lacking the carbon-19 methylene group foundin all natural vitamin D metabolites (Slatopolsky et al., Am J. KidneyDis. 26: 852 (1995)); Doxercalciferol (1α-hydroxyvitamin D2), likealfacalcidol (1α-hydroxyvitamin D3), is a prodrug which is hydroxylatedin the liver to 1α,25(OH)2D2, however, unlike alfacalcidol,doxercalciferol is also 24-hydroxylated to produce 1α,24(S)—(OH)2D2(Knutson et al., Biochem Pharmacol 53: 829 (1997)); Dihydrotachysterol2(DHT2), hydroxylated in vivo to 25(OH)DHT2, 1,25(OH)2DHT2 (McIntyre etal., Kidney Int. 55: 500 (1999)), ED-71, and eldecalcitol. See alsoErben and Musculoskel, Neuron Interact. 2(1):59-69 (2001) and Steddon etal. Nephrol. Dial. Transplant. 16 (10): 1965-1967 (2001). The foregoingreferences are incorporated by reference in their entirety.

In another embodiment, the carrier further comprises a pharmaceuticallyacceptable scaffold moiety covalently attached to the targeting groupand the insulin peptide. The scaffold moiety of the carriers of theinvention does not necessarily participate in but may contribute to thefunction or improve the pharmacokinetic properties of the insulinpeptide. The scaffolds of the invention do not substantially interferewith the binding of the targeting group to DBP. Likewise, the scaffoldsof the invention do not substantially interfere with structure orfunction of the insulin peptide. The length of the scaffold moiety isdependent upon the character of the targeting group and the insulinpeptide. One skilled in the art will recognize that various combinationsof atoms provide for variable length molecules based upon knowndistances between various bonds (Morrison, and Boyd, Organic Chemistry,3rd Ed, Allyn and Bacon, Inc., Boston, Mass. (1977), incorporated hereinby reference). Other scaffolds contemplated by the invention includepeptide linkers, protein linkers such as human serum albumin orimmunoglobulin family proteins or fragments thereof, nucleic acidlinkers, small carbon chain linkers, carbon linkers with oxygen ornitrogen interspersed, or combinations thereof. In preferredembodiments, the linkers are non-releasable or stable.

The invention comprises insulin, a therapeutic peptide. The term peptideis meant to include a string of amino acids. The amino acids in thepeptides of the invention may be naturally-occurring ornon-naturally-occurring. The peptides of the invention may besynthesized chemically or biologically, and can include cysteine-richpeptides, circular peptides, stapled peptides, peptides that include D-or L-amino acids and mixtures thereof, peptidomimetics, peptide-nucleicacids (PNAs), and combinations thereof. The invention contemplatessynthetic insulin analogs that would be improved as clinical productsthrough further modification by the methods described herein. In someembodiments, the insulin peptides of the invention are or have at leasta 90% sequence identity to SEQ ID NO: 1, 2, or 5-8.

The invention contemplates branched or cyclic insulin derivatives.Cyclic, branched and branched circular peptides result frompost-translational natural processes and are also made by suitablesynthetic methods. In some embodiments, any peptide product describedherein comprises a peptide analog described above that is thencovalently attached to an alkyl-glycoside surfactant moiety.

Other embodiments include insulin peptide chains that are comprised ofnatural and unnatural amino acids or analogs of natural amino acids. Asused herein, peptide and/or protein “analogs” comprise non-natural aminoacids based on natural amino acids, such as tyrosine analogs, whichincludes para-substituted tyrosines, ortho-substituted tyrosines, andmeta-substituted tyrosines, wherein the substituent on the tyrosinecomprises an acetyl group, a benzoyl group, an amino group, a hydrazine,an hydroxyamine, a thiol group, a carboxy group, a methyl group, anisopropyl group, a C2-C20 straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a halogen, a nitro group, or the like.

Additional embodiments include insulin peptide chains having modifiedamino acids. Examples include acylated amino acids at the ε-position oflysine, amino acids with fatty acids such as octanoic, decanoic,dodecanoic, tetradecanoic, hexadecanoic, octadecanoic, 3-phenylpropanoicacids and the like, or with saturated or unsaturated alkyl chains(Zhang, L. and Bulaj, G (2012) Curr Med Chem 19: 1602-1618, incorporatedherein by reference in its entirety).

The invention further contemplates insulin peptide chains comprisingnatural and unnatural amino acids or analogs of natural amino acids. Insome embodiments, peptide or protein “analogs” comprise non-naturalamino acids based on natural amino acids, such as tyrosine analogs,which includes para-substituted tyrosines, ortho-substituted tyrosines,and meta-substituted tyrosines, wherein the substituent on the tyrosinecomprises an acetyl group, a benzoyl group, an amino group, a hydrazine,an hydroxyamine, a thiol group, a carboxy group, a methyl group, anisopropyl group, a C2-C20 straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a halogen, a nitro group, or the like. Examples of tyrosineanalogs include 2,4-dimethyl-tyrosine (Dmt), 2,4-diethyl-tyrosine,O-4-allyl-tyrosine, 4-propyl-tyrosine, Ca-methyl-tyrosine and the like.Examples of lysine analogs include ornithine (Orn), homo-lysine,Ca-methyl-lysine (CMeLys), and the like. Examples of phenylalanineanalogs include, but are not limited to, meta-substitutedphenylalanines, wherein the substituent comprises a methoxy group, aC1-C20 alkyl group, for example a methyl group, an allyl group, anacetyl group, or the like. Specific examples include, but are notlimited to, 2,4,6-trimethyl-L-phenylalanine (Tmp), O-methyl-tyrosine,3-(2-naphthyl)alanine (Nal(2)), 3-(1-naphthyl)alanine (Nal(1)),3-methyl-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic), fluorinated phenylalanines, isopropyl-phenylalanine,p-azido-phenylalanine, p-acyl-phenylalanine, p-benzoyl-phenylalanine,p-iodo-phenylalanine, p-bromophenylalanine, p-amino-phenylalanine, andisopropyl-phenylalanine, and the like.

Also contemplated within the scope of embodiments are insulin peptidechains containing nonstandard or unnatural amino acids known to the art,for example, C-alpha-disubstituted amino acids such as Aib,Ca-diethylglycine (Deg), aminocyclopentane-1-carboxylic acid (Ac4c),aminocyclopentane-1-carboxylic acid (Ac5c), and the like. Such aminoacids frequently lead to a restrained structure, often biased toward analpha helical structure (Kaul, R. and Balaram, P. (1999) Bioorg Med Chem7: 105-117, incorporated herein by reference in its entirety).Additional examples of such unnatural amino acids useful in analogdesign are homo-arginine (Har) and the like. Substitution of reducedamide bonds in certain instances leads to improved protection fromenzymatic destruction or alters receptor binding. By way of example,incorporation of a Tic-Phe dipeptide unit with a reduced amide bondbetween the residues (designated as Tic-F[CH2-NH]^-Phe) reducesenzymatic degradation.

In some embodiments, modifications at the amino or carboxyl terminus mayoptionally be introduced into the present peptides or proteins (Nestor,J. J., Jr. (2009) Current Medicinal Chemistry 16: 4399-4418). Forexample, the present peptides or proteins can be truncated or acylatedon the N-terminus (Gourlet, P., et al. (1998) Eur J Pharmacol 354:105-111, Gozes, I. and Furman, S. (2003) Curr Pharm Des 9: 483-494), thecontents of which is incorporated herein by reference in theirentirety). Other modifications to the N-terminus of peptides orproteins, such as deletions or incorporation of D-amino acids such asD-Phe result in potent and long acting agonists or antagonists whensubstituted with the modifications described herein such as long chainalkyl glycosides.

Thus, the invention provides insulin peptide analogs wherein the nativeinsulin peptide is modified by acetylation, acylation, PEGylation,ADP-ribosylation, amidation, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-link formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, (Nestor, J. J., Jr. (2007) Comprehensive Medicinal ChemistryII 2: 573-601, Nestor, J. J., Jr. (2009) Current Medicinal Chemistry 16:4399-4418, Uy, R. and Wold, F. (1977) Science 198:890-6, Seifter, S. andEnglard, S. (1990) Methods Enzymol 182: 626-646, Rattan, S. I., et al.(1992) Ann NY Acad Sci 663: 48-62). The foregoing references areincorporated by reference in their entirety.

Glycosylated insulin peptides may be prepared using conventional Fmocchemistry and solid phase peptide synthesis techniques, e.g., on resin,where the desired protected glycoamino acids are prepared prior topeptide synthesis and then introduced into the peptide chain at thedesired position during peptide synthesis. Thus, the insulin peptidepolymer conjugates may be conjugated in vitro. The glycosylation mayoccur before deprotection. Preparation of amino acid glycosides isdescribed in U.S. Pat. No. 5,767,254, WO 2005/097158, and Doores, K., etal., Chem. Commun., 1401-1403, 2006, which are incorporated herein byreference in their entirety. For example, alpha and beta selectiveglycosylations of serine and threonine residues are carried out usingthe Koenigs-Knorr reaction and Lemieux's in situ anomerizationmethodology with Schiff base intermediates. Deprotection of the Schiffbase glycoside is then carried out using mildly acidic conditions orhydrogenolysis. A composition, comprising a glycosylated insulin peptideconjugate is made by stepwise solid phase peptide synthesis involvingcontacting a growing peptide chain with protected amino acids in astepwise manner, wherein at least one of the protected amino acids isglycosylated, followed by water-soluble polymer conjugation. Suchcompositions may have a purity of at least 95%, at least 97%, or atleast 98%, of a single species of the glycosylated and conjugatedinsulin peptide.

Monosaccharides that may by used for introduction at one or more aminoacid residues of the insulin peptides, defined and/or disclosed herein,include glucose (dextrose), fructose, galactose, and ribose. Additionalmonosaccharides suitable for use include glyceraldehydes,dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose,xylose, ribulose, xylulose, allose, altrose, mannose, N-Acetylneuraminicacid, fucose, N-Acetylgalactosamine, and N-Acetylglucosamine, as well asothers. Glycosides, such as mono-, di-, and trisaccharides for use inmodifying an insulin peptide, one or more amino acid residues of theinsulin peptides defined and/or disclosed herein include sucrose,lactose, maltose, trehalose, melibiose, and cellobiose, among others.Trisaccharides include acarbose, raffinose, and melezitose.

In further embodiments of the invention, the insulin peptides definedand/or disclosed herein may be chemically coupled to biotin. Thebiotin/insulin peptide can then bind to avidin.

Some aspects of the assembly of carriers utilizes chemical methods thatare well-known in the art. For example, Vitamin E-PEG is manufactured byEastman Chemical, Biotin-PEG is manufactured by many PEG manufacturerssuch as Enzon, Nektar and NOF Corporation. Methods of producing PEGmolecules with some vitamins and other therapeutic peptides linked tothem follow these and other chemical methods known in the art. Theattachment of PEG to an oligonucleotide or related molecule occurs, forexample, as the PEG2-N-hydroxysuccinimide ester coupled to theoligonucleotide through the 5′ amine moiety. Several coupling methodsare contemplated and include, for example, NHS coupling to amine groupssuch as a lysine residue on a peptide, maleimide coupling to sulfhydrylgroup such as on a cysteine residue, iodoacetyl coupling to a sulfhydrylgroup, pyridyldithiol coupling to a sulfhydryl group, hydrazide forcoupling to a carbohydrate group, aldehyde for coupling to theN-terminus, or tetrafluorophenyl ester coupling that is known to reactwith primary or secondary amines. Other possible chemical couplingmethods are known to those skilled in the art and can be substituted. Byway of example, conjugation using the coupling groups of the inventionmay be carried out using the compositions and methods described inWO93/012145 (Atassi et al.) and also see U.S. Pat. No. 7,803,777(Defrees et al.), incorporated by reference herein in their entirety.

Exemplary drug formulations of the invention include aqueous solutions,organic solutions, powder formulations, solid formulations and a mixedphase formulations.

Pharmaceutical compositions of this invention comprise any of thecompounds of the present invention, and pharmaceutically acceptablesalts thereof, with any pharmaceutically acceptable carrier, adjuvant orvehicle. Pharmaceutically acceptable carriers, adjuvants and vehiclesthat may be used in the pharmaceutical compositions of this inventioninclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium tri silicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Pharmaceutically acceptable salts retain the desired biological activityof the insulin composition without toxic side effects. Examples of suchsalts are (a) acid addition salts formed with inorganic acids, forexample, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid and the like/and salts formed with organic acids suchas, for example, acetic acid, trifluoroacetic acid, tartaric acid,succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid,malic acid, ascorbic acid, benzoic acid, tanic acid, pamoic acid,alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid and the like; (b) base additionsalts or complexes formed with polyvalent metal cations such as zinc,calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel,cadmium, and the like; or with an organic cation formed fromN,N′-dibenzylethylenediamine or ethlenediamine; or (c) combinations of(a) and (b), e.g. a zinc tannate salt and the like.

The pharmaceutical compositions of this invention may be administered bysubcutaneous, transdermal, oral, parenteral, inhalation, ocular,topical, rectal, nasal, buccal (including sublingual), vaginal, orimplanted reservoir modes. The pharmaceutical compositions of thisinvention may contain any conventional, non-toxic,pharmaceutically-acceptable carriers, adjuvants or vehicles. The termparenteral as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intrasynovial, intrasternal,intrathecal, intralesional, and intracranial injection or infusiontechniques.

Also contemplated, in some embodiments, are pharmaceutical compositionscomprising insulin and analogs or metabolites thereof as describedherein, or pharmaceutically acceptable salt thereof, in a mixture with apharmaceutically acceptable, non-toxic component. As mentioned above,such compositions may be prepared for parenteral administration,particularly in the form of liquid solutions or suspensions; for oral orbuccal administration, particularly in the form of tablets or capsules;for intranasal administration, particularly in the form of powders,nasal drops, evaporating solutions or aerosols; for inhalation,particularly in the form of liquid solutions or dry powders withexcipients, defined broadly; for transdermal administration,particularly in the form of a skin patch or microneedle patch; and forrectal or vaginal administration, particularly in the form of asuppository.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example, as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa.(1985), incorporated herein by reference in its entirety. Formulationsfor parenteral administration may contain as excipients sterile water orsaline alkylene glycols such as propylene glycol, polyalkylene glycolssuch as polyethylene glycol, saccharides, oils of vegetable origin,hydrogenated napthalenes, serum albumin or other nanoparticles (as usedin Abraxane™, American Pharmaceutical Partners, Inc. Schaumburg, Ill.),and the like. For oral administration, the formulation can be enhancedby the addition of bile salts or acylcarnitines. Formulations for nasaladministration may be solid or solutions in evaporating solvents such ashydrofluorocarbons, and may contain excipients for stabilization, forexample, saccharides, surfactants, submicron anhydrous alpha-lactose ordextran, or may be aqueous or oily solutions for use in the form ofnasal drops or metered spray. For buccal administration, typicalexcipients include sugars, calcium stearate, magnesium stearate,pregelatinated starch, and the like.

Delivery of modified insulin peptides described herein to a subject overprolonged periods of time, for example, for periods of one week to oneyear, may be accomplished by a single administration of a controlledrelease system containing sufficient active ingredient for the desiredrelease period. Various controlled release systems, such as monolithicor reservoir-type microcapsules, depot implants, polymeric hydrogels,osmotic pumps, vesicles, micelles, liposomes, transdermal patches,iontophoretic devices and alternative injectable dosage forms may beutilized for this purpose. Localization at the site to which delivery ofthe active ingredient is desired is an additional feature of somecontrolled release devices, which may prove beneficial in the treatmentof certain disorders.

In certain embodiments for transdermal administration, delivery acrossthe barrier of the skin would be enhanced using electrodes (e.g.iontophoresis), electroporation, or the application of short,high-voltage electrical pulses to the skin, radiofrequencies, ultrasound(e.g. sonophoresis), microprojections (e.g. microneedles), jetinjectors, thermal ablation, magnetophoresis, lasers, velocity, orphotomechanical waves. The drug can be included in single-layerdrug-in-adhesive, multi-layer drug-in-adhesive, reservoir, matrix, orvapor style patches, or could utilize patchless technology. Deliveryacross the barrier of the skin could also be enhanced usingencapsulation, a skin lipid fluidizer, or a hollow or solidmicrostructured transdermal system (MTS, such as that manufactured by3M), jet injectors. Additives to the formulation to aid in the passageof insulin peptides through the skin include prodrugs, chemicals,surfactants, cell penetrating peptides, permeation enhancers,encapsulation technologies, enzymes, enzyme inhibitors, gels,nanoparticles and peptide or protein chaperones.

One form of controlled-release formulation contains the insulin peptideor its salt dispersed or encapsulated in a slowly degrading, non-toxic,non-antigenic polymer such as copoly(lactic/glycolic) acid, as describedin the pioneering work of Kent et al., U.S. Pat. No. 4,675,189,incorporated by reference herein. The peptides may also be formulated incholesterol or other lipid matrix pellets, or silastomer matriximplants. Additional slow release, depot implant or injectableformulations will be apparent to the skilled artisan. See, for example,Sustained and Controlled Release Drug Delivery Systems, J R Robinsoned., Marcel Dekker Inc., New York, 1978; and Controlled Release ofBiologically Active Agents, R W Baker, John Wiley & Sons, New York,1987. The foregoing are incorporated by reference in their entirety.

An additional form of controlled-release formulation comprises asolution of biodegradable polymer, such as copoly(lactic/glycolic acid)or block copolymers of lactic acid and PEG, is a bioacceptable solvent,which is injected subcutaneously or intramuscularly to achieve a depotformulation. Mixing of the insulin peptides described herein with such apolymeric formulation is suitable to achieve very long duration ofaction formulations.

When formulated for nasal administration, the absorption across thenasal mucous membrane may be further enhanced by surfactants, such as,for example, glycocholic acid, cholic acid, taurocholic acid, ethocholicacid, deoxycholic acid, chenodeoxycholic acid, dehdryocholic acid,glycodeoxycholic acid, cycledextrins and the like in an amount in therange of between about 0.1 and 15 weight percent, between about 0.5 and4 weight percent, or about 2 weight percent. An additional class ofabsorption enhancers reported to exhibit greater efficacy with decreasedirritation is the class of alkyl maltosides, such as tetradecylmaltoside(Arnold, J J et al., 2004, J Pharm Sci 93: 2205-13; Ahsan, F et al.,2001, Pharm Res 18:1742-46) and references therein, all of which arehereby incorporated by reference.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant such as Ph. Helv or a similar alcohol.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers that are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are administered orally, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient that is solid at roomtemperature but liquid at the rectal temperature and therefore will meltin the rectum to release the active components. Such materials include,but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Formulations for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petroleum, white petroleum, propyleneglycol, polyoxyethylene polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical composition can be formulatedwith a suitable lotion or cream containing the active compound suspendedor dissolved in another formulation. Suitable formulations include, butare not limited to, mineral oil, sorbitan monostearate, polysorbate 60,cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol andwater. The pharmaceutical compositions of this invention may also betopically applied to the lower intestinal tract by rectal suppositoryformulation or in a suitable enema formulation. Topical transdermalpatches are also included in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When formulated for delivery by inhalation, a number of formulationsoffer advantages. Adsorption of the insulin peptide to readily dispersedsolids such as diketopiperazines (for example, Technosphere particles(Pfutzner, A and Forst, T, 2005, Expert Opin Drug Deliv 2:1097-1106)) orsimilar structures gives a formulation that results in rapid initialuptake of the insulin peptide. Lyophilized powders, especially glassyparticles, containing the insulin peptide and an excipient are usefulfor delivery to the lung with good bioavailability, for example, seeExubera® (inhaled insulin by Pfizer).

Dosage levels of between about 0.01 and about 100 mg/kg body weight perday, preferably 0.5 and about 50 mg/kg body weight per day of the activeingredient compound are useful in the prevention and treatment ofdisease. Such administration can be used as a chronic or acute therapy.The amount of drug that may be combined with the carrier to produce asingle dosage form will vary depending upon the host treated and theparticular mode of administration. A typical preparation will containfrom about 5% to about 95% active compound (w/w). Preferably, suchpreparations contain from about 20% to about 80% active compound.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, gender, diet, time of administration,rate of excretion, drug combination, the severity and course of aninfection, the patient's disposition to the infection and the judgmentof the treating physician.

The carrier-drug conjugates described herein provide advantages to drugmanufacturers and patients over unmodified drugs. Specifically, thecarrier-drug conjugate or formulation will be a more potent, longerlasting, and require smaller and less frequent dosing. This translatesinto lowered healthcare costs and more convenient drug administrationschedules for patients. The carrier-drug conjugates can also providesubcutaneous or transdermal routes of administration as alternatives tointravenous injection. These routes can be self-administered by patientsand thus improve patient compliance.

In yet another aspect of the invention, the levels of DBP can beincreased as part of the carrier-drug therapy. It has been reported thatestrogen can increase DBP levels (Speeckaert et al., Clinica ChimicaActa 371:33). It is contemplated here that levels of DBP can beincreased by administration of estrogen for more effective delivery ofcarrier-drug conjugates.

In yet another aspect of the invention, it is contemplated that thecarrier can be used to deliver drugs transdermally. Since DBP normallytransports UV activated vitamin D at locations close to the surface ofthe skin, the use of a transdermal delivery system with the carrierbecomes feasible.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner. Inparticular, the compositions and methods disclosed herein function withall non-hormonal forms of vitamin D, including homologs, analogs, andmetabolites thereof. This includes vitamin D3 as used in the examplesbelow.

EXAMPLES Example 1 Preparation of Exemplary Carriers for CouplingInsulin Peptides to Non-Hormonal Vitamin D at the C25 Position

Exemplary carriers were prepared containing vitamin D and 2 kDa PEGscaffolds. One exemplary carrier was thiol-reactive and comprisedvitamin D-PEG with a maleimide reactive group at the C25 position.Another exemplary carrier was amine-reactive and comprised vitamin D-PEGwith an NHS-reactive group. These reagents were prepared as described inWO2013172967 (Soliman et al.), incorporated herein by reference in itsentirety.

Example 2 Preparation of an Exemplary Amino-terminal Reactive Carrierfor Coupling Insulin Peptides to Non-Hormonal Vitamin D at the C3Position

An exemplary amino-terminal reactive carrier was prepared containing analdehyde reactive group connected to the C3 position of vitamin D and a2 kDa PEG scaffold (VitD-(3)-PEG_(2k)-aldehyde). The aldehyde on thecarrier in this example was used to conjugate to a free amino-terminuson the proteins and peptides disclosed in the examples below. Thesynthesis is outlined in FIG. 1.

Briefly,(S,Z)-3-((E)-2-((1R,3aS,7aR)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methylhexahydro-1H-inden-4(2H)-ylidene)ethylidene)-4-methylenecyclohexanol(compound Va, 20 mg, 0.049 mmol, 1 equiv.) purchased from TorontoResearch Chemicals, catalog number C125700, also known as calcifedioland 25-hydroxyvitamin D) was dissolved in a mixture of anhydroustert-butanol and acetonitrile (10:1, 1 mL), cooled to 4° C.Acrylonitrile (26.6 mg, 0.5 mmol, 10 equiv., was added to it followed byTriton B, 40% aqueous solution, 10 μL). The mixture was stirred at 4° C.for 2.5 h. The reaction was quenched with cold 2% HCl (10 mL), theaqueous phase was extracted with ether (2×10 mL), dried (MgSO₄) andevaporated to obtain the crude product. This material was purified byflash chromatography (TLC, silica gel, 50% ethyl acetate in hexanes)with 5-20% EtOAc/hexanes as eluent to isolate the desired product,3-(((S,Z)-3-((E)-2-((1R,3aS,7aR)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methylhexahydro-1H-inden-4(2H)-ylidene)ethylidene)-4-methylenecyclohexyl)oxy)propanenitrile,compound Vc (15 mg, 68%) as a white solid (R_(f) 0.2 silica gel, 40%EtOAc in hexanes). NMR analysis did not show any appreciable amount ofsolvents.

To a solution of aluminum chloride (66 mg, 0.495 mmol) in anhydrousether (2 mL) at 0° C. under argon was added a solution of lithiumaluminum hydride (1M in ether, 19 mg, 0.5 mL, 0.5 mmol) dropwise. Themixture was stirred for 5 min., a solution of compound Vc (15 mg, 0.033mmol) in ether (3 mL) was added to it dropwise, the reaction mixture wasstirred at 0° C. for 5 min and then at room temperature for 1 h. Thereaction was monitored by MS and TLC (silica gel, 10% MeOH/CHCl₃/0.1%NH₄OH). Ethyl acetate (1 mL) and water (1 mL) were added to the reactionmixture followed by 5% NaOH (5 mL). The organic phase was separated, andthe aqueous phase was extracted with ethyl acetate (5 mL) and ether (5mL). The combined organic phases were washed with brine (5 mL), dried(Na₂SO₄) and evaporated on a rotavap to afford the desired amine,(R)-6-((1R,3aS,7aR,E)-4-((Z)-2-((S)-5-(3-aminopropoxy)-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-yl)-2-methylheptan-2-ol,compound Vd (12.5 mg, 82%) as a pale yellow oil. R_(f) 0.2 (silica gel,20% MeOH/DCM/0.2% NH₄OH). The NMR analysis revealed the presence ˜8% ofethyl acetate.

Compound Vd (12.5 mg, 0.0273 mmol, 1 equiv.), compound Ve (hydroxyl PEGNHS ester, MW 2000 with n≅45 where n is the number of repeating CH₂CH₂Ounits, Jenkem Technology USA #A-5076, 43 mg, 0.0216 mmol, 0.8 equiv.)were dissolved in anhydrous dichloromethane (0.1 mL). Triethylamine (12mg, 16 μl, 0.11 mmol, 4 equiv.) was added and the reaction mixture wasstirred for 20 h at room temperature under nitrogen. The sample wasdried under a stream of nitrogen to afford the crude compound Vf, whichwas purified by flash chromatography using 5-10% MeOH/dichloromethane aseluent to isolate the desired product Vf as a white foam (30 mg, 38%).R_(f) 0.4 (silica gel, 10% methanol in dichloromethane). ¹H NMR analysisof the isolated material confirmed its identity and purity.

To a solution of compound Vf (30 mg, 0.0123 mmol, 1 equiv.),tetrapropylammonium perruthenate (1.0 mg, 0.00284, 0.23 equiv.) andN-methylmorpholine-N-Oxide (4.3 mg, 0.0369 mmol, 3 equiv.) in 2 mL ofdry dichloromethane was added powdered 4A° molecular sieves (500 mg) andthe reaction mixture was flushed with N₂. The reaction flask was coveredwith aluminum foil to avoid light and it was stirred at room temperaturefor 36 h. Since the R_(f) of both starting material and product is sameon TLC (silicagel, 10% MeOH/dichloromethane), formation of the productwas confirmed by examining the ¹HNMR of an aliquot. The reaction mixturewas filtered through the pad of Celite in a pipette with dichloromethane(15 mL) and N₂ pressure. The combined organics were concentrated under aflow of N₂ and dried on high vacuum for 2 h to get 35 mg (100%) of thecrude product TLC (R_(f): 0.3, 10% MeOH/dichloromethane, staining withPMA). A second run of reaction under the exactly same conditions yieldedanother 35 mg of the product. ¹H NMR of the product from both batches issame and hence combined to get 70 mg of compound V,VitD-(3)-PEG_(2k)-aldehyde.

Example 3 Preparation of an Exemplary Thiol-Reactive Carrier forCoupling Insulin Peptides to Non-Hormonal Vitamin D at the C3 Position

An exemplary thiol-reactive carrier comprising vitamin D with amaleimide reactive group connected to the C3 position of vitamin D(VitD-(3)-PEG_(2k)-maleimide) was prepared. The maleimide on the carrierin this example was used to conjugate to a free thiol on the protein andpeptide in the examples below. The synthesis is outlined in FIG. 2.

Briefly, compound Vd (23 mg, 0.05 mmol, 1 equiv.) prepared as in Example2, compound VIa (Creative Pegworks cat. # PHB-956, MAL-PEG-COOH, 2 kwith n≅45 where n is the number of repeating CH₂CH₂O units, 79 mg,0.0395 mmol, 0.8 equiv.) and 2-chloro-1-methylpyridinium iodide (32 mg,0.125 mmol, 2.5 equiv.) were dissolved in anhydrous dichloromethane (1mL). Triethylamine (20.4 mg, 28 μl, 0.2 mmol, 4 equiv.) was added andthe reaction mixture was stirred for 4 h at room temperature undernitrogen. The reaction mixture was diluted with dichloromethane (20 mL),washed with 5% aqueous citric acid (20 mL), saturated aqueous sodiumbicarbonate (20 mL), and brine (20 mL). The organic layer was dried overanhydrous sodium sulfate, filtered and concentrated at 30° C. The samplewas purified by silica gel (10 g) flash chromatography. The column waseluted with 1-10% MeOH/dichloromethane. Fractions containing pureproduct were combined together and evaporated on a rotavap, whilemaintaining the temperature at 30° C. The sample was dried under astream of nitrogen to afford compound VI, VitD-(3)-PEG_(2k)-maleimide asa brown gum (58 mg, 48%) (R_(f) 0.25, silica gel, 10% methanol indichloromethane)¹H NMR analysis of the isolated material confirmed itsidentity and purity.

Example 4 Preparation of an Exemplary Amine-Reactive Carrier forCoupling Insulin Peptides to Non-Hormonal Vitamin D at the C3 Position

An exemplary amine-reactive carrier comprising vitamin D with an NHSreactive group connected to the C3 position of vitamin D(VitD-(3)-PEG_(1.3k)-NHS) was prepared. The NHS on the carrier in thisexample was used to conjugate to a free thiol on the protein and peptidein the examples below. The synthesis is outlined in FIG. 3.

Briefly, compound Vd (20 mg, 0.044 mmol, 1 equiv.) and compound VIIa(Quanta Biodesign cat. #10140, with n=25 where n is the number ofrepeating CH₂CH₂O units, 44 mg, 0.0346 mmol, 0.8 equiv.) were dissolvedin anhydrous dichloromethane (1 mL). Triethylamine (22.0 mg, 31 μl, 0.22mmol, 5 equiv.) was added and the reaction mixture was stirred for 24 hat room temperature under nitrogen. The reaction mixture was dilutedwith dichloromethane (20 mL), washed with 5% aqueous citric acid (20mL), and brine (20 mL). The organic layer was dried over anhydroussodium sulfate, filtered and concentrated while maintaining thetemperature at 30° C. The sample was purified by silica gel (10 g) flashchromatography. The column was eluted with 1-10% MeOH/dichloromethane.Fractions containing pure product were combined together and evaporatedon a rotavap, while maintaining the temperature below 30° C. The samplewas dried under a stream of nitrogen to afford compound VIIb as a browngum (33 mg, 56%) (R_(f) 0.20, silica gel, 10% methanol indichloromethane). ¹H NMR analysis of the isolated material confirmed itsidentity.

Compound VIIb (31 mg, 0.018 mmol, 1 equiv.), N-hydroxysuccinimide (6.3mg, 0.055 mmol, 3 equiv.), and EDCI (8.6 mg, 0.045 mmol, 2.5 eq.) weredissolved in anhydrous THF (2 mL). Triethylamine (7.4 mg, 10 μL, 0.073mmol, 4 equiv.) was added and the reaction mixture was stirred for 24 hat room temperature under nitrogen. The reaction mixture was dilutedwith dichloromethane (20 mL) and washed with 5% aqueous citric acid (20mL), and brine (20 mL). The organic layer was dried over anhydroussodium sulfate, filtered and concentrated while maintaining thetemperature at 30° C. The sample was dried under a stream of nitrogen toafford compound VII, VitD-(3)-PEG_(1.3k)-NHS, as a brown gum (38.6mg, >100%) (R_(f) 0.25, silica gel, 10% methanol in dichloromethane). ¹HNMR analysis of the isolated material confirmed its identity and purity.

Example 5 Preparation of Insulin Coupled to Non-Hormonal Vitamin D atthe C25 and C3 Positions

In this example, the VitD-(25)-PEG_(2k)-NHS was conjugated to humaninsulin comprising the A chain (SEQ ID NO:1) and B chain (SEQ ID NO:2)to prepare a therapeutic for treating diabetes. The insulin A chaincontains a cys6-cys11 intra-chain disulfide linkage. The cys7 on the Achain is linked to cys7 of the B chain by an inter-chain disulfidelinkage. The cys20 on the A chain is linked to cys19 of the B chain,also by an inter-chain disulfide linkage. The NHS reactive group targetsthe carrier to amino reactive side chains on residues such as lysinewhen reacted as described below. Thus, this reaction in this exampletargeted lysine 29 on the insulin B-chain. Insulin (Sigma Aldrich, St.Louis, Mo., Catalog #I2643) was resuspended in a 1:1 mixture of DMSO and1M HEPES+0.85% NaCl, pH=8 at a concentration of 5 mg/ml.VitD-(25)-PEG_(2k)-NHS carrier dissolved in DMSO at a concentration of 5mg/ml 1.4 to 4 molar equivalents relative to insulin was added. Thefinal concentration of insulin was brought to1 mg/ml in dH₂O and thereaction was allowed to proceed for 1 hour at room temperature. Theinsulin conjugates were confirmed by SDS-PAGE.

In this example, the VitD-(3)-PEG_(1.3k)-NHS is conjugated to humaninsulin to prepare a therapeutic for treating diabetes. Insulin (SigmaAldrich, St. Louis, Mo., Catalog #I2643) is resuspended in a 1:1 mixtureof DMSO and 1M HEPES+0.85% NaCl, pH=8 at a concentration of 5 mg/ml.VitD-(2)-PEG_(1.3k)-NHS carrier is dissolved in DMSO at a concentrationof 5 mg/ml 1.4 to 4 molar equivalents relative to insulin was added. Thefinal concentration of insulin is brought to 1 mg/ml in dH₂O and thereaction is allowed to proceed for 1 hour at room temperature. Theinsulin conjugates are confirmed by SDS-PAGE.

Pharmacokinetic experiments, in vitro bioactivity assays measuring theuptake of glucose by adipocytes, and evaluation in vivo of the bloodglucose lowering ability in diabetic rat models are performed asdescribed in EP2085406, incorporated herein by reference in itsentirety.

Example 6 Preparation of Insulin Coupled to Non-Hormonal Vitamin D atthe C25 and C3 Positions

Synthesis of VitD-(25)-PEG_(2K)-Insulin

In this example, the VitD-(25)-PEG_(2K)-NHS from Example 1 wasconjugated to human insulin comprising the A chain (SEQ ID NO:1) and Bchain (SEQ ID NO:2) to prepare a therapeutic for treating diabetes. A 2KDa PEG scaffold was conjugated to the carbon 25 atom on the vitamin Dmolecule. This carrier was conjugated to lysine 29 on the insulinB-chain. Insulin (Sigma Aldrich, St. Louis, Mo., Catalog #I2643) wasresuspended in a 95:5 mixture of DMSO and triethylamine (TEA) at aconcentration of 10 mg/ml. VitD-(25)-PEG_(2K)-NHS carrier was dissolvedin a 95:5 mixture of DMSO and TEA at a concentration of 10 mg/ml.1.3-1.6 molar equivalents of the VitD-(25)-PEG_(2k)-NHS carrier wasadded to 1.0 molar equivalents of insulin and the reaction was allowedto proceed at room temperature for 2-16 hours in the dark. Two volumesof deionized water were added to the reaction mixture, and the pH wasadjusted to between 8 and 9 by the addition of 2N HCl. SDS-PAGE analysisshowed that insulin conjugated to one VitD-(25)-PEG_(2K) carrier was themajor product but unmodified insulin and insulin conjugated to twocarriers were also present. The desired insulin conjugate with onecarrier was purified by anion exchange chromatography (HiTrap Q HPcolumn, GE Healthcare) in buffer A (20 mM Tris pH=8.5, 50% ethanol) witha 20 minute gradient to 60% buffer B (20 mM Tris pH=8.5, 1 M NaCl, 50%ethanol). The insulin conjugate was confirmed by SDS-PAGE and MALDI-TOFmass spectrometry.

Synthesis of VitD-(3)-PEG

In this example, the VitD-(3)-PEG_(1.3K)-NHS (VII) from Example 4 wasconjugated to human insulin at lysine 29 on the B-chain to prepare atherapeutic for treating diabetes. As a result of the conjugationreaction, the scaffold was a 1.2 kDa PEG. It was attached to vitamin Don the 3 position. The reaction was performed in a similar manner asdescribed above for the VitD-(25)-PEG_(2K)-NHS carrier. The desiredinsulin conjugate with one VitD-(3)-PEG_(1.2K)-carrier was purified byanion exchange chromatography (HiTrap Q HP column, GE Healthcare) inbuffer A (20 mM Tris pH=8.5, 50% methanol) with a 20 minute gradient to30% buffer B (20 mM Tris pH=8.5, 1 M NaCl, 50% methanol). The insulinconjugate was confirmed by SDS-PAGE analysis.

Synthesis of 20K-PEG-Insulin

In this example, 20 kD PEG-NHS was conjugated to human insulin toprepare a benchmark therapeutic for treating diabetes (see EP2288375).The reaction was performed in a similar fashion as described above,except 0.7 molar equivalents of methoxy PEG succinimidyl carboxymethylester, MW 20000 (Jenkem Cat. No. M-SCM-20K) dissolved in 95:5acetonitrile: TEA at a concentration of 100 mg/ml was reacted with 1.0molar equivalent of insulin dissolved in 95:5 DMSO:TEA at aconcentration of 10 mg/ml. The desired insulin conjugate with one 20 kDPEG conjugate was purified by anion exchange chromatography (HiTrap Q HPcolumn, GE Healthcare) in buffer A (20 mM Tris pH=8.5) with a 15 minutegradient to 100% buffer B (20 mM Tris pH=8.5, 1 M NaCl). The insulinconjugate was confirmed by SDS-PAGE analysis.

Activity of Insulin Constructs in Cell-Based INSRb Receptor Assay:

Unmodified insulin, 20K-PEG-insulin, VitD-(25)-PEG_(2K)-insulin, andVitD-(3)-PEG_(1.2K)-insulin were analyzed using the PathHunter® U2OSINSRb Functional Assay (Discover RX, Inc., Freemont, Calif., Cat. No.93-0466C3). U-2 OS cells expressing the insulin receptor, isoform B,measures the activation of this receptor by recruiting an SH2 fusionprotein that is dependent on receptor phosphorylation. This leads tocomplementation of two fragments of the beta-galactosidase enzyme. FIG.4 compares the functional activity of insulin vs. the three modifiedpeptides. The curves were fit with a four parameter logistic function inorder to determine the EC₅₀ values (Table 1). The results show that allthe compounds have activity against the insulin receptor INSRb in thisassay.

TABLE 1 Compound EC₅₀ Insulin  7.9 ng/ml 20K-PEG-insulin 26.3 ng/mlVitD-(25)-PEG_(2K)-insulin 15.8 ng/ml VitD-(3)-PEG_(1.2K)-insulin 282.2ng/ml 

It was observed that the smaller PEG size in VitD-(3)-PEG_(1.2K)-insulinresulted in a lower solubility in solution and the cell-based assay thanthe insulin conjugates with 2 kDa or 20 kDa PEG. This molecule, however,showed a higher solubility in serum that likely resulted from DBPbinding. For example, when VitD-(3)-PEG_(1.2K)-insulin was diluted inPBS buffer, quantitation by ELISA yielded a four-fold lower value thanwhen a similar dilution was performed in either serum or a Tween20-containing buffer. As discussed below, this conjugate showed the bestpharmacokinetic profile and glucose reduction in vivo.

Pharmacokinetic and Pharmacodynamics Properties of Insulin and InsulinConjugates:

Groups of three rats each were injected either intravenously orsubcutaneously with insulin, VitD-(25)-PEG_(2K)-insulin,20K-PEG-insulin, or VitD-(3)-PEG_(1.2K)-insulin at a dose of 0.02 mg/kg(0.58 IU/kg) (iv) or 0.04 mg/kg (1.15 IU/kg) (sc). Plasma samples weretaken at 5 minutes (iv only), 0.5, 1, 2, 4, 6, 8, 24, 32, 48, and 56hours and analyzed for the quantity of insulin and glucose. Insulin wasmeasured by the SPI-BIO Insulin (mouse/rat) EIA kit (Cayman Chemicals,Ann Arbor, Mich. Cat. No. 589501). All of the modified insulinderivatives had improved pharmacokinetic profiles when compared tonative insulin (FIGS. 5A and 5B). Unmodified insulin decayed tonear-background levels within 15-30 minutes of iv injection and 45-60minutes of sc injection. The 20K-PEG-insulin andVitD-(25)-PEG_(2K)-insulin improved the in vivo half life. TheVitD-(3)-PEG_(1.2K)-insulin, however, showed the most dramaticpharmacokinetic properties. This demonstrates that conjugation of thecarrier to the C3 position of vitamin D is preferred to conjugation atthe C25 position. Also, VitD-(3)-PEG_(1.2K)-insulin was superior to20K-PEG-insulin. This demonstrated that the vitamin D moiety provides asignificant benefit in extending the half-life of insulin.

VitD-(3)-PEG_(1.2K)-insulin was highly effective at stably reducingblood glucose levels in vivo. Blood glucose levels were determined inrats injected with free glucose and the conjugates using the Amplex RedGlucose/Glucose Oxidase Assay Kit (Invitrogen, Carlsbad, Calif., Cat.No. A22189). FIG. 6 shows that free insulin caused a sharp drop inglucose levels lasting 30 minutes. In contrast,VitD-(3)-PEG_(1.2k)-insulin and VitD-(25)-PEG_(2K)-insulin stablyreduced blood glucose levels for approximately eight hours.VitD-(3)-PEG_(2K)-insulin caused a larger and more sustained drop inglucose levels than VitD-(25)-PEG_(2K)-insulin. The PEG20K-insulinconjugate did not display significant glucose lowering ability for anextended time.

Exemplary Sequences

(human insulin A Chain) SEQ ID NO: 1 GIVEQCCTSICSLYQLENYCN (human insulin B Chain)  SEQ ID NO: 2: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (Vitamin D Binding Protein (DBP))  SEQ ID NO: 3MKRVLVLLLAVAFGHALERGRDYEKNKVCKEFSHLGKEDFTSLSLVLYSRKFPSGTFEQVSQFVKEVVSFTEACCAEGADPDCYDTRTSAFSAKSCESNSPFPVHPGTAECCTKEGFERKLCMAALKHQPQEFPTYVEPTNDEICEAFRKDPKEYANQFMWEYSTNYGQAPLSLLVSYTKSYLSMVGSCCTSASPTVCFLKERLQLKHLSLLTTLSNRVCSQYAAYGEKKSRLSNLIKLAQKVPTADLEDVLPLAEDITNILSKCCESASEDCMAKELPEHTVKLCDNLSTKNSKFEDCCQEKTAMDVFVCTYFMPAAQLPELPDVELPTNKDVCDPGNTKVMDKYTFELSRRTHLPEVFLSKVLEPTLKSLGECCDVEDSTTCFNAKGPLLKKELSSFIDKGQELCADYSENTFTEYKKKLAERLKAKLPDATPTELAKLVNKHSDFASNCCSINSPPLYCDSEIDAELKNIL  (Vitamin D Binding Protein (DBP)) SEQ ID NO: 4 TTTAATAATAATTCTGTGTTGCTTCTGAGATTAATAATTGATTAATTCATAGTCAGGAATCTTTGTAAAAAGGAAACCAATTACTTTTGGCTACCACTTTTACATGGTCACCTACAGGAGAGAGGAGGTGCTGCAAGACTCTCTGGTAGAAAAATGAAGAGGGTCCTGGTACTACTGCTTGCTGTGGCATTTGGACATGCTTTAGAGAGAGGCCGGGATTATGAAAAGAATAAAGTCTGCAAGGAATTCTCCCATCTGGGAAAGGAGGACTTCACATCTCTGTCACTAGTCCTGTACAGTAGAAAATTTCCCAGTGGCACGTTTGAACAGGTCAGCCAACTTGTGAAGGAAGTTGTCTCCTTGACCGAAGCCTGCTGTGCGGAAGGGGCTGACCCTGACTGCTATGACACCAGGACCTCAGCACTGTCTGCCAAGTCCTGTGAAAGTAATTCTCCATTCCCCGTTCACCCAGGCACTGCTGAGTGCTGCACCAAAGAGGGCCTGGAACGAAAGCTCTGCATGGCTGCTCTGAAACACCAGCCACAGGAATTCCCTACCTACGTGGAACCCACAAATGATGAAATCTGTGAGGCGTTCAGGAAAGATCCAAAGGAATATGCTAATCAATTTATGTGGGAATATTCCACTAATTACGGACAAGCTCCTCTGTCACTTTTAGTCAGTTACACCAAGAGTTATCTTTCTATGGTAGGGTCCTGCTGTACCTCTGCAAGCCCAACTGTATGCTTTTTGAAAGAGAGACTCCAGCTTAAACATTTATCACTTCTCACCACTCTGTCAAATAGAGTCTGCTCACAATATGCTGCTTATGGGGAGAAGAAATCAAGGCTCAGCAATCTCATAAAGTTAGCCCAAAAAGTGCCTACTGCTGATCTGGAGGATGTTTTGCCACTAGCTGAAGATATTACTAACATCCTCTCCAAATGCTGTGAGTCTGCCTCTGAAGATTGCATGGCCAAAGAGCTGCCTGAACACACAGTAAAACTCTGTGACAATTTATCCACAAAGAATTCTAAGTTTGAAGACTGTTGTCAAGAAAAAACAGCCATGGACGTTTTTGTGTGCACTTACTTCATGCCAGCTGCCCAACTCCCCGAGCTTCCAGATGTAGAGTTGCCCACAAACAAAGATGTGTGTGATCCAGGAAACACCAAAGTCATGGATAAGTATACATTTGAACTAAGCAGAAGGACTCATCTTCCGGAAGTATTCCTCAGTAAGGTACTTGAGCCAACCCTAAAAAGCCTTGGTGAATGCTGTGATGTTGAAGACTCAACTACCTGTTTTAATGCTAAGGGCCCTCTACTAAAGAAGGAACTATCTTCTTTCATTGACAAGGGACAAGAACTATGTGCAGATTATTCAGAAAATACATTTACTGAGTACAAGAAAAAACTGGCAGAGCGACTAAAAGCAAAATTGCCTGATGCCACACCCACGGAACTGGCAAAGCTGGTTAACAAGCACTCAGACTTTGCCTCCAACTGCTGTTCCATAAACTCACCTCCTCTTTACTGTGATTCAGAGATTGATGCTGAATTGAAGAATATCCTGTAGTCCTGAAGCATGTTTATTAACTTTGACCAGAGTTGGAGCCACCCAGGGGAATGATCTCTGATGACCTAACCTAAGCAAAACCACTGAGCTTCTGGGAAGACAACTAGGATACTTTCTACTTTTTCTAGCTACAATATCTTCATACAATGACAAGTATGATGATTTGCTATCAAAATAAATTGAAATATAATGCAAACCATAAAAAAAAAAAAAAAAAAAAAA  A(Insulin analog A chain sequence 3)  SEQ ID NO: 5 GIVEQCCTSICSLYQLENYCG (Insulin analog B chain sequence 1)  SEQ ID NO: 6FVNQHLCGSHLVEALYLVCGERGFFYTKPT  (Insulin analog B chain sequence 2)SEQ ID NO: 7 FVNQHLCGSHLVEALYLVCGERGFFYTPDT (Insulin analog B chain sequence 3)  SEQ ID NO: 8FVNQHLCGSHLVEALYLVCGERGFFYTPKTKK 

All publications and patent documents disclosed or referred to hereinare incorporated by reference in their entirety. The foregoingdescription has been presented only for purposes of illustration anddescription. This description is not intended to limit the invention tothe precise form disclosed. It is intended that the scope of theinvention be defined by the claims appended hereto.

What is claimed:
 1. A method of treating a patient in need of insulin,comprising administering an effective amount of a pharmaceuticalcomposition comprising said insulin conjugated to the carbon 3 positionof a non-hormonal vitamin D targeting group that is not hydroxylated atthe carbon 1 position.
 2. The method of claim 1, wherein saidpharmaceutical composition is delivered to said patient by atransdermal, oral, parenteral, subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intrasynovial, intrasternal,intrathecal, intralesional, intracranial injection, infusion,inhalation, ocular, topical, rectal, nasal, buccal, sublingual, vaginal,or implanted reservoir mode.
 3. A method of manufacturing thepharmaceutical composition of claim 1, comprising conjugating saidtargeting group and said insulin, wherein said conjugating step utilizesa coupling group.
 4. The method according to claim 3, wherein saidcoupling group is selected from the group consisting of anamine-reactive group, a thiol-reactive group, a maleimide group, a thiolgroup, an aldehyde group, an NETS-ester group, a haloacetyl group, aniodoacetyl group, a bromoacetyl groups, a SMCC group, a sulfo SMCCgroup, a carbodiimide group, bifunctional cross-linkers, NHS-maleimido,and combinations thereof.
 5. The pharmaceutical composition resultingfrom the method of claim 3, wherein said composition comprises acarrier-drug compound containing a linkage selected from the groupconsisting of a thiol linkage, an amide linkage, an oxime linkage, ahydrazone linkage, and a thiazolidinone linkage.
 6. The method accordingto claim 3, wherein said conjugating step is accomplished bycycloaddition reactions.
 7. A pharmaceutical carrier comprising aformula I:B-(L)-^(a) S-(M)-^(b)-C  I Wherein: B is a targeting group that is avitamin D that is not hydroxylated at the carbon 1 position, conjugatedat the carbon 3 position to (L)^(a) ; S is a scaffold moiety, comprisingpoly(ethylene glycol), polylysine, polyethyleneimine,poly(propyleneglycol), a peptide, an amino acid, a nucleic acid, aglycan, a modifying group that contains a reactive linker, polylacticacid, a water-soluble polymer, a small carbon chain linker, or anadditional therapeutic moiety; C is an amine-reactive group, athiol-reactive group, a maleimide group, a thiol group, a disulfidegroup, an aldehyde group, an NETS-ester group, a 4-nitrophenyl ester, anacylimidazole, a haloacetyl group, an iodoacetyl group, a bromoacetylgroups, a SMCC group, a sulfo SMCC group, a carbodiimide group andbifunctional cross-linkers such as NHS-Maleimido or combinationsthereof; (L)^(a) and (M)^(b) are linkers independently selected from—(CH₂)_(n)—, —C(O)NH—, —HNC(O)—, —C(O)O—, —OC(O)—, —O—, —S—S—, —S—,—S(O)—, —S(O)₂— and —NH—; a is an integer from 0-4; b is an integer from0-4; and n is an integer from 0-3.
 8. The pharmaceutical carrier ofclaim 7 comprising formula V:


9. The pharmaceutical carrier of claim 7 comprising formula VI:


10. The pharmaceutical carrier of claim 7 comprising formula VII:


11. The carrier-drug conjugate of claim 5, wherein said insulin peptideretains substantially the same activity as said insulin peptide notconjugated to said targeting group as measured by a functional assay.12. The method of claim 1, wherein said insulin comprises a firstpeptide having an amino acid sequence with at least a 90% sequenceidentity to SEQ ID NO:1 and a second peptide having an amino acidsequence with at least a 90% sequence identity to SEQ ID NO:2.
 13. Themethod of claim 12, wherein said first peptide has the amino acidsequence of SEQ ID NO:1.
 14. The method of claim 12, wherein said firstpeptide has the amino acid sequence of SEQ ID NO:5.
 15. The method ofclaim 12, wherein said second peptide has the amino acid sequence of SEQID NO:2.
 16. The method of claim 12, wherein said second peptide has theamino acid sequence of SEQ ID NO:6.
 17. The method of claim 12, whereinsaid second peptide has the amino acid sequence of SEQ ID NO:7.
 18. Themethod of claim 12, wherein said second peptide has the amino acidsequence of SEQ ID NO:8.
 19. The method of claim 3, wherein saidtargeting group is conjugated to said insulin peptide via a scaffoldthat is selected from the group consisting of poly(ethylene glycol),polylysine, polyethyleneimine, poly(propyleneglycol), a peptide, serumalbumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid,a glycan, a modifying group that contains a reactive linker, awater-soluble polymer, a small carbon chain linker, and an additionaltherapeutic peptide.
 20. The method of claim 19, wherein said scaffoldis approximately the same mass as the insulin peptide.